JP6660103B2 - Immunoassay for detecting neurotoxic amino acids associated with neuropathy - Google Patents

Description

RELATED APPLICATIONS This application claims the benefit of priority to US Provisional Application No. 61 / 079,334, filed July 9, 2008, which is expressly incorporated herein by reference in its entirety. It is.

  The present invention relates to antibodies that bind to β-N-methylamino-L-alanine (BMAA), and immunoassays and kits for screening a sample to detect the presence of BMAA in the sample.

  The non-protein amino acid β-N-methylamino-L-alanine (BMAA) is produced by various taxa of cyanobacteria [1] and is administered in vivo and in vitro under various experimental conditions. It has been shown to have neuroexcitatory and neurotoxic effects. Because BMAA can be found in powders formed from Cycad species, BMAA is considered a candidate neurotoxin associated with a unique neurological disease identified several decades ago among the Chamorros of Guam. Is a combination of symptoms that have clinical similarities to the features of amyotrophic lateral sclerosis (ALS), parkinsonism, and dementia, so that amyotrophic lateral sclerosis in Guam-Parkinson's dementia It is known as disease complex (ALS-PDC), which is thought to be related to dietary habits, including BMAA-containing cycad flour (among others, Non-Patent Documents 2 and 3). Bioaccumulation of BMAA in the food chain, for example, in Guam, BMAA produced by cyanobacterial symbionts in cycad roots is taken up by cycad hosts, and fruit bats (bats) that further accumulate BMAA in their tissues Fruit bats (bats), which are stored in structures such as the sarcotesta of the seeds and the gametophytes of the seeds, and which are eaten by people, and which have a high accumulation level of BMAA, can be dramatic when eaten by people. It has been shown to have bioaccumulation (among others, Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6).

  In recent years, BMAA has been detected in the tissues of subjects who do not eat cycads or fruit bats, and some subjects with detectable levels of BMAA have found that Alzheimer's disease, ALS, and progressive supranuclear palsy ( A neurological disorder such as PSP) has been clinically diagnosed (based on symptoms) or has been confirmed (eg, based on autopsy of brain tissue) while having detectable levels of BMAA Other subjects had asymptomatic stages of neuropathy (Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8, Patent Document 1, Patent Document 2, Patent Document 3).

  A chromatographic method for analyzing tissue and environmental samples for neurotoxic amino acids such as BMAA by high performance liquid chromatography (HPLC) or HPLC mass spectrometry (HPLC-MS) analysis of the tissue and / or environmental samples. It is disclosed (Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8, Patent Document 1, Patent Document 2).

U.S. Patent No. 7,256,002 US Patent Application Publication No. 2007/0254315 U.S. Patent Application Publication No. 2007/0292993

Cox et al. (2005) Proc Natl Acad Sci USA 102: 5074-5078. Spencer et al. (1987) Science 237: 517-522. Kisby et al. (1992) Neurogeneration 1: 73-82. Bannack et al. (2003) Neurology 61387-389. Cox et al. (2002) Neurology 58: 956-959. Cox et al. (2003) Proc Natl Acad Sci USA 100: 13380-13383. Murch et al. (2004) Proc Natl Acad Sci USA 101: 12228-12231. Murch et al. (2004) Acta Neurol Scan 110: 267-269.

  The present invention provides an immunoassay for screening a sample to detect the presence of β-N-methylamino-L-alanine (BMAA). The invention provides an immunoassay, wherein free BMAA is detected, or protein-bound BMAA is detected, or both free BMAA and protein-bound BMAA are detected. The immunoassay provided herein can be an enzyme-linked immunosorbent assay (ELISA), which can be, but is not limited to, an antibody capture assay, an indirect competitive ELISA, or a direct ELISA. The immunoassay provided herein can be an immunoblot assay.

  The invention provides an immunoassay for screening a sample to detect the presence of BMAA, wherein the immunoassay includes an antibody that binds to BMAA. The invention provides an immunoassay for screening a sample to detect the presence of BMAA, which comprises binding to BMAA, L-alanine, L-glutamine, L-tyrosine, glycyl-glycine, L- Antibodies that do not substantially bind to an amino acid selected from the group consisting of glycine, L-leucine, L-phenylalanine, gamma-aminobutyric acid (GABA), L-glutamic acid, and L-aspartic acid. The invention provides an immunoassay comprising an antibody that binds to BMAA, wherein the antibody can be a polyclonal, monoclonal, or antibody fragment. The present invention provides an immunoassay comprising an antibody that binds to BMAA, wherein the antibody can be detectably labeled, wherein the label is a radioactive label, a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a colloidal gold label, a dye moiety, It can be, but is not limited to, a magnetic compound, a detectable enzyme, biotin, avidin, or streptavidin. The invention provides an immunoassay comprising an antibody that binds to BMAA, wherein the antibody can be an antibody that is not detectably labeled, wherein the immunoassay comprises a detectably labeled secondary antibody that binds to an unlabeled antibody that binds to BMAA. Further comprising an antibody, wherein the label can be a radioactive label, a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a colloidal gold label, a dye moiety, a detectable enzyme, a detectable ligand, biotin, avidin, or streptavidin, It is not limited to these. Secondary antibodies can be labeled with horseradish peroxidase (HRP).

  The present invention provides an immunoassay for screening a sample to detect the presence of BMAA, wherein the immunoassay further comprises an amplification step.

  The present invention screens a sample to detect the presence of BMAA using an immunoassay as provided herein by contacting the sample with an antibody that binds BMAA and detecting the antibody. Provide a way to: The invention provides a method for screening a tissue sample from a subject to detect the presence of BMAA in a tissue sample using an immunoassay, as provided herein, wherein the method comprises the steps of: The presence of a detectable amount of BMAA indicates the subject's exposure to an environmental source of BMAA. The present invention provides a method for screening a sample to detect the presence of BMAA in a tissue sample, including, but not limited to, neural or non-neural tissue, wherein the neural tissue comprises hair, It can be keratinous tissue such as skin, nails, claws, or hooves. The present invention provides a method for screening a sample to detect the presence of BMAA using an immunoassay as provided herein by detecting protein-bound BMAA on an immunoblot of a tissue sample. I will provide a. The present invention provides a method for screening an environmental sample for detecting the presence of BMAA in the environmental sample using an immunoassay as provided herein, wherein the environmental sample comprises a water sample, Alternatively, it can be, but is not limited to, a sample from a foodstuff. The present invention provides a method for screening an environmental sample to detect the presence of BMAA in the environmental sample using an immunoassay as provided herein, the method comprising: The method can further include screening the sample to detect a cyanobacterial substance. The present invention provides a method for screening environmental samples by detecting the presence of protein-bound BMAA on the immunoblot of the environmental sample and further detecting cyanobacterial proteins on the immunoblot.

  The present invention provides antibodies that bind to BMAA. The present invention relates to BMAA, which binds to L-alanine, L-glutamine, L-tyrosine, glycyl-glycine, L-glycine, L-leucine, L-phenylalanine, gamma-aminobutyric acid (GABA), L-glutamic acid, and An antibody is provided that does not substantially bind to an amino acid selected from the group consisting of L-aspartic acid. The invention provides an antibody that binds to BMAA, wherein the antibody is capable of binding to free BMAA, or the antibody binds to protein-bound BMAA, or the antibody binds to both free and protein-bound BMAA. To join. The invention provides an antibody that binds to BMAA, wherein the antibody binds to the L-BMAA isomer and does not substantially bind to the D isomer of BMAA. Antibodies that bind to BMAA as described herein can be polyclonal, or monoclonal, or antibody fragments. Antibodies that bind to BMAA as described herein can be detectably labeled. Antibodies that bind BMAA as described herein can be labeled for use in in vivo imaging.

The present invention provides a kit for screening a sample to detect the presence of BMAA, wherein the kit is compartmentalized to receive one or more container means (containers). And the kit comprises at least one container means with an antibody that binds to BMAA. In a kit as provided herein, the antibody that binds to BMAA can be detectably labeled, wherein the kit comprises at least one container with means for detecting the labeled antibody bound to the sample. Means (container) can be further included. In a kit as provided herein, the antibody that binds to BMAA can be unlabeled, and further comprises at least one container means with a labeled secondary antibody that binds to the unlabeled antibody. And can further comprise container means with means for detecting labeled secondary antibody binding to unlabeled antibody bound to the sample. Kits as provided herein can include container means with a control sample containing a known amount of BMAA. Kits as provided herein can include means for preparing a sample to detect the presence of BMAA, including such means for mechanically separating the sample. And means for chemically separating the sample, but are not limited to these. The invention provides a kit for screening a tissue sample from a subject to detect the presence of BMAA, wherein the tissue sample can be, but is not limited to, a keratinous tissue sample, such as hair or skin. . The present invention provides kits for screening environmental samples to detect the presence of BMAA, which may be, but is not limited to, a water sample or a sample from a foodstuff. Kits as provided herein can contain means for screening a plurality of sample types.
In a preferred embodiment of the present invention, for example, the following is provided:
(Item 1)
Immunoassay for screening samples to detect the presence of β-N-methylamino-L-alanine (BMAA).
(Item 2)
2. The immunoassay according to item 1, wherein free BMAA is detected.
(Item 3)
2. The immunoassay according to item 1, wherein protein-bound BMAA is detected.
(Item 4)
2. The immunoassay according to item 1, wherein both free BMAA and protein-bound BMAA are detected.
(Item 5)
2. The immunoassay according to item 1, wherein the immunoassay is an enzyme-linked immunosorbent assay (ELISA).
(Item 6)
6. The immunoassay according to item 5, wherein the ELISA is an antibody capture assay.
(Item 7)
6. The immunoassay according to item 5, wherein the ELISA is an indirect competitive ELISA.
(Item 7)
6. The immunoassay according to item 5, wherein the ELISA is a direct ELISA.
(Item 8)
2. The immunoassay according to item 1, wherein the immunoassay is an immunoblot assay. (Item 10)
2. The immunoassay according to item 1, comprising an antibody that binds to BMAA.
(Item 11)
The antibodies that bind to BMAA are L-alanine, L-glutamine, L-tyrosine, glycyl-glycine, L-glycine, L-leucine, L-phenylalanine, gamma-aminobutyric acid (GABA), L-glutamic acid, and L-glutamic acid. -The immunoassay according to item 10, wherein the immunoassay does not substantially bind to an amino acid selected from the group consisting of aspartic acid.
(Item 12)
The immunoassay according to item 10, wherein the antibody that binds to BMAA is a polyclonal antibody.
(Item 13)
Item 11. The immunoassay according to item 10, wherein the antibody that binds to BMAA is a monoclonal antibody.
(Item 14)
Item 11. The immunoassay according to item 10, wherein the antibody that binds to BMAA is an antibody fragment.
(Item 15)
11. The immunoassay according to item 10, wherein the antibody that binds to BMAA is detectably labeled.
(Item 16)
The antibody uses a label selected from the group consisting of a radioactive label, a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a colloidal gold label, a dye moiety, a paramagnetic compound, a detectable enzyme, biotin, avidin, and streptavidin. 16. The immunoassay according to item 15, which is labeled with:
(Item 17)
11. The immunoassay of item 10, wherein the antibody that binds to BMAA is not detectably labeled, and further comprises a detectably labeled secondary antibody that binds to the antibody that binds to BMAA. (Item 18)
The secondary antibody is a group consisting of a radiolabel, a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a colloidal gold label, a dye moiety, a paramagnetic compound, a detectable enzyme, a detectable ligand, biotin, avidin, and streptavidin. Item 18. The immunoassay according to Item 17, wherein the immunoassay is labeled with a label selected from:
(Item 19)
19. The immunoassay according to item 18, wherein the secondary antibody is labeled with a detectable enzyme.
(Item 20)
20. The immunoassay according to item 19, wherein the detectable enzyme is horseradish peroxidase (HRP).
(Item 21)
2. The immunoassay according to item 1, further comprising an amplification step.
(Item 22)
A method for screening a sample to detect the presence of β-N-methylamino-L-alanine (BMAA) according to the immunoassay according to item 1, comprising contacting an antibody that binds to BMAA with the sample. And detecting the antibody.
(Item 23)
Screening a tissue sample from the subject to detect the presence of BMAA in the tissue sample, wherein the presence of a detectable amount of BMAA in the sample is indicative of exposing the subject to an environmental source of BMAA. 23. The method according to item 22, comprising:
(Item 24)
24. The method according to item 23, wherein the tissue sample is a nerve tissue.
(Item 25)
24. The method according to item 23, wherein the tissue sample is non-neural tissue.
(Item 26)
26. The method according to item 25, wherein the non-neural tissue is keratinous tissue.
(Item 28)
27. The method according to item 26, wherein the keratinous tissue is hair.
(Item 29)
27. The method according to item 26, wherein the keratinous tissue is skin.
(Item 30)
27. The method according to item 26, wherein the keratinous tissue is a nail, a claw, or a hoof.
(Item 31)
24. The method of item 23, comprising detecting the presence of protein-bound BMAA on an immunoblot of the tissue sample.
(Item 32)
23. The method according to item 22, wherein the sample is an environmental sample.
(Item 33)
33. The method according to item 32, wherein the environmental sample is a water sample.
(Item 34)
33. The method according to item 32, wherein the environmental sample is from a food product.
(Item 35)
33. The method according to item 32, further comprising screening the sample for detecting cyanobacterial substances in the sample.
(Item 36)
33. The method of item 32, comprising detecting the presence of protein-bound BMAA on an immunoblot of the environmental sample, further comprising detecting cyanobacterial proteins on the immunoblot.
(Item 37)
An antibody that binds to BMAA.
(Item 38)
Selected from the group consisting of L-alanine, L-glutamine, L-tyrosine, glycyl-glycine, L-glycine, L-leucine, L-phenylalanine, gamma-aminobutyric acid (GABA), L-glutamic acid, and L-aspartic acid 39. The antibody of item 37, wherein the antibody does not substantially bind to the amino acid to be made.
(Item 39)
38. The antibody according to item 37, wherein the antibody binds to free BMAA.
(Item 40)
38. The antibody according to item 37, wherein the antibody binds to protein-bound BMAA.
(Item 41)
38. The antibody according to item 37, wherein the antibody binds to both free BMAA and protein-bound BMAA.
(Item 42)
38. The antibody of item 37, wherein the antibody binds to the L-BMAA isomer and does not substantially bind to the D isomer of BMAA.
(Item 43)
38. The antibody according to item 37, wherein the antibody is a polyclonal antibody.
(Item 44)
38. The antibody according to item 37, wherein the antibody is a monoclonal antibody.
(Item 45)
38. The antibody according to item 37, wherein the antibody is an antibody fragment.
(Item 46)
38. The antibody according to item 37, wherein the antibody is detectably labeled.
(Item 47)
38. The antibody according to item 37, wherein the antibody is labeled for use in an in vivo diagnostic imaging method.
(Item 48)
A kit for screening a sample to detect the presence of β-N-methylamino-L-alanine (BMAA), comprising a delivery means compartmented to receive one or more container means. Wherein the at least one container means comprises an antibody that binds to BMAA.
(Item 49)
49. The kit according to item 48, wherein the antibody that binds to BMAA is detectably labeled.
(Item 50)
50. The kit of claim 49, further comprising at least one container means, comprising means for detecting the labeled antibody bound to the sample.
(Item 51)
49. The kit according to item 48, wherein the antibody that binds to BMAA is not labeled.
(Item 52)
52. The kit of claim 51, further comprising container means comprising a labeled secondary antibody that binds to said unlabeled antibody.
(Item 53)
53. The kit of claim 52, further comprising container means comprising means for detecting the labeled secondary antibody binding to the unlabeled antibody bound to the sample.
(Item 54)
49. The kit according to item 48, further comprising container means comprising a control sample containing a known amount of BMAA.
(Item 55)
Item 4 further comprising means for preparing said sample to detect the presence of BMAA
9. The kit according to 8.
(Item 56)
56. The kit according to item 55, wherein said means for preparing said sample comprises means for mechanically separating said sample.
(Item 57)
56. The kit according to item 55, wherein said means for preparing said sample comprises means for chemically separating said sample.
(Item 58)
49. The kit according to item 48, for screening a tissue sample from a subject.
(Item 59)
59. The kit according to item 58, wherein the tissue sample is a keratin tissue sample.
(Item 60)
49. The kit according to item 48, for screening an environmental sample.
(Item 61)
61. The kit according to item 60, wherein the environmental sample is a water sample.
(Item 62)
61. The kit according to item 60, wherein the environmental sample is from a foodstuff.
(Item 63)
49. A kit according to item 48 for screening a plurality of sample types.

Figure 2 outlines the conjugation and immunization procedures used to generate and test antibodies to BMAA, where KLH is keyhole limpet hemocyanin, BSA is bovine serum albumin, and GLU is Glutaraldehyde and EDC is carbodiimide. On the MAXISORP ™ (FIG. 3A), MEDISORP ™ (FIG. 3B), and MULTISORP ™ (FIG. 3C) plates in the presence of buffers having different pH values as indicated, BMAA Measures the reactivity of antisera (EDC1-1, EDC2-1, Glu1, Glu2, Glu3, Glu4) and null serum (NS1, NS2) produced against the BMAA complex at a coating concentration of 20 μg / mL. 5 shows the results from an antibody capture immunoassay for From left to right, BMAA, L-alanine (L-Ala), L-glutamine (L-Gln), L-glutamine linked with glutaraldehyde at coating concentrations of 0.2 mM, 0.5 mM, 1 mM, and 10 mM. Tyrosine (L-Tyr), glycyl-glycine (glygly), L-glycine (L-Gly), L-leucine (L-leu), L-phenylalanine (L-Phe), gamma-aminobutyric acid (GABA), L -Using glutamate (L-Glu) and L-aspartate (L-Asp) at a dilution of 1/1000 (panel A) and a dilution of 1/2000 (panel B), the KLH-GLU of the BMAA complex -Shows the results from an antibody capture immunoassay to measure the reactivity of the antiserum raised against BMAA (third anti-KGB bleed). 5 shows images of immunoblots of BSA-BMAA complexes probed with antisera raised against KLH-BMAA complexes, wherein lane 1 of each blot is BSA-GLU-BMAA (BGB) , Lane 2 of each blot contains BSA-EDC-BMAA (BEB), lane 3 of each blot contains native BSA, and blot A contains anti-KGB antiserum at a dilution of 1/100. Blot B was probed with anti-KGB antiserum at a dilution of 1/200, Blot C was probed with anti-KGB antiserum at a dilution of 1/500, and Blot D was probed with Blot E was probed with the anti-KEB antiserum at a dilution of 1/200, and Blot F was probed with the anti-KEB antiserum at a dilution of 1/100. It is probed with anti-KEB antiserum at a dilution of 0. Shown is an image of an immunoblot of a total protein extract of Cylindrospermopsis raciborskii strain CR3 ("CR3 protein") probed with antisera raised against the BMAA complex, wherein: Lane 1 of each blot contains BSA, lane 2 of each blot contains CR3 protein, lane 3 of each blot contains CR3 protein pre-incubated with BMAA, and blot A Blot B was probed with anti-KGB antiserum at a dilution of 1/200, and Blot C was probed with anti-KGB antiserum at a dilution of 1/200. Blot D was anti-KE at 1/100 dilution. B was probed with antiserum, Blot E was probed with anti-KEB antiserum at a dilution of 1/200, and Blot F was probed with anti-KEB antiserum at a dilution of 1/500. Shown is an image of an immunoblot of a whole protein extract of Kirindrospermopsis rakiborskii strain CR3 ("CR3 protein") and a parallel BSA control probed with antisera raised against the BMAA complex, Lanes contain native BSA, even lanes contain CR3 total protein, lanes 3-10 are probed with anti-KGB antiserum, lanes 12-10 are with anti-KEB antiserum. Probed, lanes 2 and 12 were probed with null serum as follows. Lane 2, CR3 protein probed with null serum at 1/200 dilution; Lane 3, BSA probed with anti-KGB antiserum at 1/200 dilution; Lane 4, 1/200 dilution. CR3 protein probed with anti-KGB antiserum; lane 5, BSA probed with anti-KGB antiserum at 1/500 dilution; lane 6, with anti-KGB antiserum at 1/500 dilution. Lane 7, probed with anti-KGB antiserum at 1/1000 dilution; lane 8, CR3 protein probed with anti-KGB antiserum at 1/1000 dilution; lane 9, BSA probed with anti-KGB antiserum at 1/2000 dilution; lane 10, using anti-KGB antiserum at 1/2000 dilution Lane 12, CR3 protein probed with null serum at 1/200 dilution; lane 13, BSA probed with anti-KEB antiserum at 1/200 dilution; lane 14 , CR3 protein probed with anti-KEB antiserum at 1/200 dilution; lane 15, BSA probed with anti-KEB antiserum at 1/500 dilution; lane 16, at 1/500 dilution. CR3 protein probed with anti-KEB antiserum; lane 17, BSA probed with anti-KEB antiserum at 1/1000 dilution; lane 18, with anti-KEB antiserum at 1/1000 dilution. Probed CR3 protein; lane 19, BSA probed with anti-KEB antiserum at 1/2000 dilution And Lane 20, 1/2000 CR3 protein probed with anti-KEB antiserum at a dilution of. Kirindrospermopsis rakiborskii strain CR3 ("CR3 protein"), E. coli strain HK29 ("E. coli strain HK29 protein"), Chlorella vulgaris probed with antisera raised against the BMAA complex. ) (“Chlorella protein”), and immunoblot images of total protein extracts (20 μg protein per lane) of pure protein of Tetraselmis protein (“Tetraselmis protein”). , Lanes 1 and 11 contain molecular weight markers (100-1000 Da), lanes 2, 6, 12, and 16 contain CR3 protein, and lanes 3, 7, 13, and 17 contain E. coli HK29 protein. Contain Lanes 4, 8, 14, and 18 contain chlorella protein, lanes 5, 9, 15, and 19 contain tetracermis protein, and lanes 2-5 show null serum at 1/500 dilution. Lanes 6-9 are probed with anti-KEB antiserum at a dilution of 1/500, lanes 12-15 are probed with anti-KEB antiserum at a dilution of 1/1000, Lanes 16-19 were probed with anti-KGB antiserum at a dilution of 1/500.

  The present disclosure provides immunoassays, antibodies, and kits for screening samples to detect neurotoxic amino acids associated with a neurological disorder. The present invention provides immunoassays, antibodies, and kits for detecting the presence of BMAA in a sample, comprising contacting the sample with an antibody that binds BMAA and detecting the antibody bound to the sample. Including. Immunoassays, antibodies, and kits are provided for screening samples to detect β-N-methylamino-L-alanine (BMAA) in the sample. Provided are immunoassays, antibodies, and kits for screening subjects to detect exposure of BMAA to environmental sources. Provided are immunoassays, antibodies, and kits for screening environmental samples to identify environmental sources of BMAA.

  The present invention provides an immunoassay, an antibody, and a method for detecting the presence of BMAA in a tissue sample from a subject by contacting the tissue sample with an antibody that binds to BMAA and detecting the antibody bound to the tissue sample. And kits. According to one aspect of the present invention, the immunoassay of the invention is used to detect BMAA in a tissue sample from a subject, and the immunoassay, the antibody, and the antibody are used to screen a subject for exposure to an environmental source of BMAA. And a kit is provided, wherein the presence of a detectable amount of BMAA in the tissue sample is such that the subject is exposed to an environmental source of BMAA because BMAA has been accumulated to detectable levels in the tissue being screened. Indicates that it is. In accordance with another aspect of the present invention, the subject uses the immunoassay of the present invention to detect BMAA in a tissue sample from the subject for neurological disorders, particularly exposure to neurotoxic amino acids associated with BMAA. Immunoassays, antibodies, and kits are provided for screening for the presence of BMAA in a tissue sample, indicating that the subject has been exposed to a neurotoxic amino acid associated with a neurological disorder. . According to another aspect of the present invention, a subject suffering from or at risk of suffering from a neurological disease is screened using the immunoassay of the present invention to detect BMAA in a tissue sample from the subject. For this purpose, immunoassays, antibodies, and kits are provided.

  The present invention provides immunoassays, antibodies, and kits for determining the presence of BMAA in an environmental sample by contacting an antibody that binds to BMAA with the environmental sample and detecting the antibody bound to the environmental sample. I do. According to one aspect of the present invention, there is provided an immunoassay, an antibody, and a kit for screening for an environmental source of BMAA using the immunoassay of the present invention to detect BMAA in an environmental sample. The presence of a detectable amount of BMAA indicates that the sample is an environmental source of BMAA. According to another aspect, immunoassays, antibodies, and kits are provided for screening environmental samples for neurotoxic amino acids associated with a neurological disorder, particularly BMAA. In one embodiment, the invention provides an immunoassay for detecting the presence of BMAA in an environmental sample that can be or has been ingested by a subject. In one embodiment, the environmental sample may include, but is not limited to, a water sample or foodstuff.

  Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, non-limiting exemplary embodiments of the methods and materials are described herein. It is described in.

  It is understood that the term "antibody" originally refers to a polypeptide substantially encoded by an immunoglobulin gene or fragment thereof that specifically binds and recognizes an antigen target. The term "antibody" as used in refers to immunoglobulins, immunoglobulin fragments, intact antibodies, polyclonal antibodies, monoclonal antibodies, serum responses to antigens (ie, antisera raised against antigens), endogenous genetic Antibodies produced by sequence expression, recombinant antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies (newly synthesized), multivalent antibodies, single-chain antibodies, antibody fragments, antibody subsequences, ability to bind antigen Antibody portion, monovalent Fab fragment, divalent F (ab ') 2 fragment, Fd fragment, dAb fragment Fragments, Fv fragments (variable fragments), single-chain Fv fragments (scFvs), isolated complementarity determining regions (CDRs), epitope binding polypeptides generated using phage display libraries, and the like. Can be used to describe an antibody of the invention, eg, a recombinant monoclonal antibody that binds to BMAA.

  The phrase "detecting the presence of BMAA" or "detecting the presence of BMAA", or a similar phrase or grammatical equivalent, determines the presence or absence of a detectable level or amount of BMAA in the sample. And generally includes determining or quantifying the level or amount of BMAA in the sample. It should be understood that the terms are used in a non-limiting manner. Further, it is understood that the phrase "determining BMAA level" or "determining BMAA level" or similar phrases includes determining, or quantifying, the level or amount of BMAA in a sample. I want to. In one non-limiting embodiment, an immunoassay is provided that only determines whether detectable levels of BMAA are present in a sample. In one non-limiting embodiment, an immunoassay is provided that provides a means for determining or quantifying the level or amount of BMAA in a sample or sample fraction. In other non-limiting embodiments, for example, to determine whether the level of BMAA in one sample is increased or decreased compared to the level detected in another sample, An immunoassay is provided that allows for the analysis and comparison of multiple samples.

  As used herein, the phrase "screening" or "screening," or similar phrase, refers to screening to detect neurotoxic amino acids associated with a neurological disorder. And screening to determine the level or amount of neurotoxic amino acids, including, but not limited to, the actual or potential of a subject to neuropathic amino acids associated with a neurological disorder, particularly BMAA. Also involves screening tissue samples from the subject to determine specific exposure, and screening to identify environmental samples containing neurotoxic amino acids associated with neurological disorders, particularly BMAA. .

  "Binding specificity" or "specific binding" refers to substantial recognition of a first molecule for a second molecule, and substantial binding. The present invention relates to an antiserum produced against BMAA conjugated to a carrier protein capable of substantially recognizing and binding BMAA (anti-BMAA antiserum), ie, binding specificity for BMAA. Anti-BMAA antiserum having sexual and specific binding is provided. The present invention provides an antibody having binding specificity and specific binding to BMAA, wherein the antibody of the present invention includes a polyclonal antibody, a monoclonal antibody, or an antibody fragment, for example, having binding properties to BMAA. An Fv, single chain Fv, Fab ', or F (ab') 2 fragment includes, but is not limited to.

  As used herein, the phrase "substantial binding" or "substantially binds" or similar phrases refers to specific binding or recognition between molecules in an assay mixture under certain assay conditions. Or reactive amount. As used herein, substantial binding is structurally similar to the ability of an antibody to bind or recognize BMAA (target molecule), eg, one or more different molecules, eg, BMAA (non-target molecule). With respect to the difference in the ability to bind the selected amino acid to the lack of antibody, the difference is therefore sufficient to allow a significant assay to detect BMAA to be performed under a particular set of assay conditions. Assay conditions that can affect the binding or reaction between molecules include, but are not limited to, the relative concentrations of target and non-target molecules, and the time and temperature of the incubation. Similarly, substantial binding relates to the difference between the reactivity of an antibody with BMAA and the lack of an antibody reactive with one or more different molecules, eg, amino acids structurally similar to BMAA, The differences are sufficient to allow a significant assay to detect BMAA to be performed under a particular set of assay conditions. As used herein, the phrase "substantially does not bind" or "does not substantially cross-react" generally refers to the amount of binding or recognizing between molecules in an assay mixture under particular assay conditions. An antibody capable of binding or recognizing BMAA is substantially incapable of binding or recognizing another molecule, such as a structurally similar amino acid, ie, an antibody capable of binding or recognizing BMAA is substantially Therefore, it cannot cross-react with other molecules such as structurally similar amino acids. Antibodies that are reactive with BMAA may have binding capacity, or cross-reactivity with structurally similar amino acids, under a specific set of assay conditions, including relative concentrations of molecules and incubation. It is less than 25%, preferably less than 10%, more preferably less than 5% of the reactivity shown for BMAA. Antibodies with reactivity with BMAA that “substantially do not bind” structurally similar amino acids can exhibit detectable binding to structurally similar amino acids, or under a specific set of assay conditions Cannot show detectable binding to structurally similar amino acids. Specific binding, substantial binding, or loss of substantial binding can be determined by a number of well-known methods, such as enzyme-linked immunosorbent assays (ELISAs), particularly antibody capture or indirect competitive ELISAs, or immunoblots ("Western" Blot ") assay, or radioimmunoassay (RIA), or immunohistochemical assay.

  As provided herein, a subject can be any organism suitable for performing the methods of the invention. In particular, the subject is a mammal, more particularly, a primate, and even more specifically, a human. In one embodiment, the subject is a laboratory animal that is exposed to a neurotoxic amino acid associated with a neurological disorder or a neurotoxic derivative thereof. Such experimental animals include, but are not limited to, mice, rabbits, rats, bats, pigs, sheep, cows, monkeys, apes, or other animals suitable for studying neuropathy. In one embodiment, the method of the invention is performed using an experimental animal for an animal model in which one or more neurological diseases are present. In another embodiment, the methods of the invention are performed using laboratory animals as part of developing an animal model for one or more neurological disorders. In yet another embodiment, the method of the present invention is directed to a laboratory animal wherein the effect of exposure to a neurotoxic amino acid or a neurotoxic derivative thereof associated with a neurological disorder is determined by a study of brain chemistry, structure, or function. Is performed using In one embodiment, the subject is a human. In another embodiment, the subject is a human suffering from one or more neurological disorders. In another embodiment, the subject is a human who is asymptomatic for one or more neuropathy. In another embodiment, the subject is a human who has been identified as being at risk for developing a neurological disorder. In yet another embodiment, the subject is a human known or suspected of having been exposed to at least one neurotoxic amino acid or neurotoxic derivative thereof associated with a neurological disorder.

  All publications, patents, and references cited herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control.

  As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

Assays for Neurotoxic Amino Acids in Samples According to one aspect of the present invention, immunoassays, antibodies, and kits are provided for detecting the presence of one or more forms of neurotoxic amino acids associated with a neurological disorder, particularly BMAA. You.

  According to one aspect, to detect the presence of neurotoxicity in a subject by analyzing a tissue sample from the subject to detect the presence of a neurotoxic amino acid, particularly one or more forms of BMAA, Immunoassays and kits are provided, which may indicate the presence of neurotoxicity. According to another aspect, immunoassays and kits are provided for detecting an environmental source of neurotoxicity by analyzing an environmental sample to detect the presence of a neurotoxic amino acid, particularly one or more forms of BMAA. This may indicate the presence of neurotoxicity.

  The neurotoxic amino acids, particularly BMAA, in a sample can be in a “free” form (eg, cytoplasmic, circulating, unbound, readily released), a “protein-bound” form (eg, bound to the surface of a protein, Alternatively, the "free" and "protein-bound" forms may be associated with other cellular components (e.g., saccharides, lipids, or polymers (e.g., (Conjugated to cellulose, chitin, amylose, proteoglycan), modified to other sample components (eg, carbamate adducts), derivatized, or otherwise linked. . It will be appreciated that any or all of these forms (eg, free, protein bound) may be detected by one of skill in the art and may vary depending on the immunoassay used for the detection. In one non-limiting exemplary embodiment, the BMAA can be present in a tissue (environmental) sample in free (unbound) form or in a protein-bound form, wherein the protein-bound form includes BMAA bound to the surface of a protein (for example, linked via a spacer group as a side group by a complex, covalent bond, or non-covalent bond, etc.) or BMAA incorporated into an amino acid chain forming the polypeptide backbone of the protein. But not limited to them. In one embodiment, both free BMAA levels and protein-bound BMAA levels are determined. In one embodiment, only free BMAA levels are determined. In another embodiment, only the level of protein-bound BMAA is determined. In another embodiment, all forms of BMAA present in the sample are released as "free" BMAA, such that after treating the sample, for example, by hydrolysis or digestion, the total BMAA in the sample is determined. . In certain embodiments, one of skill in the art can determine one or more BMAA complexes of interest in a sample and determine whether the immunoassay or antibody used is appropriate for detecting the BMAA complexes. And it can be determined whether the sample should be processed to make a BMAA complex that can be used for detection. In other embodiments, an immunoassay is provided for determining additional amino acids, proteins, or other components in addition to BMAA.

  The immunoassays, antibodies, and kits provided herein analyze any sample according to the present invention, including, but not limited to, tissue samples from subjects and environmental samples used in environmental screening. It should be understood that these are available to those skilled in the art. One skilled in the art will be able to prepare an environmental sample as needed to fit a particular property of the sample, e.g., to prepare a keratinous tissue sample for analysis, or to include a substance having cellulose, chitin, or proteoglycan cell walls. It is to be understood that the method of the present invention can be modified as needed to do so.

Antibodies to Neurotoxic Amino Acids and Neurotoxic Derivatives The present invention provides antibodies that bind to neurotoxic amino acids and neurotoxic derivatives thereof, and to detect the presence of at least one neurotoxic amino acid or neurotoxic derivative thereof in a sample. And methods and kits for utilizing these antibodies. According to one aspect, the invention provides antibodies that bind to BMAA, as well as methods and kits for utilizing these antibodies to detect the presence of BMAA in a tissue sample. According to one aspect, the present invention provides methods and kits for utilizing these antibodies to detect the presence of BMAA in an environmental sample. According to another aspect, the invention provides an immunoassay for determining BMAA in a tissue or environmental sample.

  As discussed above, the term “antibody” as used herein refers to an immunoglobulin, an immunoglobulin fragment, an intact antibody, a single-chain antibody, an antibody fragment, a serum reaction to an antigen (to an antigen). Antibodies produced, including naturally occurring antibodies (ie, resulting from the expression of endogenous genetic sequences), recombinant antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies (synthesized antibodies). , Polyclonal antibodies, monoclonal antibodies, Fvs (variable fragments), single-chain Fvs (scFvs), Fab ', or F (ab') 2 fragments, but are not limited thereto. Polyclonal antibodies are prepared by immunizing a host animal with the antigen, collecting the serum after immunization, and characterizing the serum with binding specificity for the antigen (antiserum to the antigen). It should be understood that this can be done. Pancreatic cells from the immunized animal are harvested using methods known in the art, and fused with, for example, myeloma cells, by immunizing the cells, and then having the desired specificity and affinity. (Kohler and Milstein, 1975, Nature 256: 495-497) can be used to prepare monoclonal antibodies, and the monoclonal antibodies can be further modified or optimized using recombinant DNA techniques. Please understand that you can do it. The term "antibody" is in its original sense of a polypeptide substantially encoded by an immunoglobulin gene or fragment thereof, for example, as described in Paul, ed. , Fundamental Immunology (3rd ed. 1993), which can refer to specifically binding and recognizing antigen targets, as described in detail. The term "antibody" includes fragments obtained from the enzymatic or chemical degradation of intact antibodies (polyclonal or monoclonal), or fragments that can be newly synthesized, by use of recombinant DNA methodology or by chemical synthesis. Antibody fragments, eg, Fv or scFv. The term "antibody" includes recombinant antibodies, including chimeric antibodies, humanized antibodies, recombinant monoclonal antibodies, and the like.

  Amino acids, certain drugs, organic compounds, metals, hypotoxins, and low molecular weight compounds such as peptides and oligosaccharides having a molecular weight of less than 2-5 kDa, when administered in the presence of an immune system stimulating adjuvant. Even they are usually not immunogenic. In order to generate an immune response to these compounds, it is necessary to conjugate such compounds to an immunogenic carrier compound such as an immunogenic carrier protein. The term hapten is generally understood to refer to lower molecular weight compounds conjugated to an immunogenic carrier compound, where in the hapten carrier structure the hapten is such that the low molecular weight compound is itself immunogenic. It should be understood that, even if not available, it can serve as an antigen. The hapten-carrier complex is then used to immunize a recipient animal (eg, a mouse, rat, sheep, goat, or rabbit) according to known methods to elicit an immune response in the recipient animal. Optional steps include adjuvant (eg, complete Freund's adjuvant, CFA) and hapten carrier complex for initial immunization, or multiple initial immunizations, and one or more “booster” immunizations. Including mixing. The products of the immune response are then collected and analyzed to identify the antibody response to the hapten.

Methods for producing antibodies using hapten-carrier conjugates are known in the art. Protocols for selecting an immunogenic carrier compound and conjugating the hapten to the immunogenic carrier compound are described, for example, in Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory (Cold Spring Harbor, NY, 1988). Known in the art as described in 78-87. Examples of the immunogenic carrier compound include thyroglobulin, β-galactosidase, dextran, polylysine, tuberculin-derived protein, ovalbumin (OVA), bovine serum albumin (BSA), sheep serum albumin, goat serum albumin, or fish serum albumin. Serum albumin, and keyhole limpet hemocyanin (KLH), but are not limited thereto. An immunogenic carrier compound can be selected that is not presumably present in the sample and can be analyzed to determine the presence of a hapten (eg, a neurotoxic amino acid or derivative thereof). This makes it possible to use antisera without having to isolate the anti-hapten antibody from the anti-carrier antibody. Its large size makes it highly immunogenic and the large number of lysine residues available for conjugation makes it very useful as a carrier for haptens, for example, absorption-system proteins found in molluscs Is often selected, while phylogenetic separation between molluscs and other taxa is analyzed to determine the presence of KLH carrier protein and neurotoxic amino acids or derivatives. Reduces the risk of cross-reactivity between antibodies to naturally occurring proteins in the sample being prepared. In one embodiment, the BMAA or BMAA derivative is conjugated to a carrier protein selected on the assumption that it does not presumably occur in a sample that can be analyzed to determine the presence of the BMAA or BMAA derivative. In one embodiment, the carrier protein is KLH. In one embodiment, the carrier protein is BSA.

  Antibodies to small molecules, such as amino acids and amino acid derivatives, are known in the art. U.S. Pat. No. 4,762,781 describes making and using antibodies to various amino-containing small molecules; U.S. Pat. No. 5,112,738 describes making and using antibodies to histamine; Geffford et al. (1985, J Neurochem 44: 1221-1228) describes making and using antisera to indolealkylamines. U.S. Patent No. 6,608,178 describes the preparation and use of an antibody specific for the seratonin metabolite 5-hydroxytryptophor (5-HTOL) to detect recent alcohol consumption. Where 5-HTOL or 5-HTOL glucuronide or sulfate complex is specific, and seratonin (5-hydroxytryptoamine, 5-HT), seratonin metabolite 5-hydroxyindole-3- Antibodies were developed that did not have specific binding activity for acetic acid (5-HIAA), and other compounds such as structurally related indoles and other glucuronides. The production and use of polyclonal antibodies to excitatory amino acids such as glutamate has been described by a number of groups, for example, Hepler et al. (1983, J Histochem Cytochem 36:13), Petrusz et al. (1990, Brain Res 529: 339), and Ordronneau et al. (1991, J Immunol Methods 142: 169-176). Both polyclonal and monoclonal antibodies to various amino acids and amino acid derivatives, such as neurotransmitters, are known in the art and include, for example, polyclonal anti-aspartate, polyclonal anti-GABA, polyclonal anti-glutamate, polyclonal anti-seratonin, monoclonal Anti-GABA, monoclonal anti-aspartate, monoclonal anti-glutamate, and the like are commercially available, and are all available from Sigma Aldrich Co. , St. Available from Louis, MO, or polyclonal antibodies to amino acids are available from Signature Immunology (Salt Lake City UT).

  Without wishing to be bound by this theory, it should be noted that haptens based on a single molecule linked to a carrier compound are almost always present in linear determinants, Molecular structure in space characterized by adjacent interaction sites with limited mobility. Linear determinants are highly restricted to the immune system such that antibodies that bind to the hapten target substantially always attack the same or overlapping linear epitopes to the extent that binding is mutually exclusive. 1 shows a series of targets that were performed. Thus, anti-hapten antibodies produced by the polyclonal method very often have monoclonal properties, as long as molecular selectivity is taken into account. This is important for at least two reasons: (A) The limiting property of a particular anti-hapten antibody is generally its affinity for its hapten target, and (b) the polyclonal method usually provides a higher affinity, selective anti-hapten antibody than the monoclonal method. Provide a quick and easy way to find out. The affinity of antibodies for non-linear determinant immunogens, such as large proteins, is complex, but very small and linear antigenic, with virtually all useful antibodies converging in a very narrow range of affinities. This should be contrasted with the case of hapten immunogens having determinants. In addition, the components of the linear determinant may have some mobility, but the linear determinant may be present in a small number of structures representing different targets, and antibodies may be generated against different target structures This operation is usually limited, as can be done.

  As provided herein, a neurotoxic amino acid or neurotoxic derivative thereof is conjugated to an immunogenic carrier compound to form an immunogenic hapten carrier complex that is administered to a recipient animal, An immune response is generated against the body, and the products of the immune response are then analyzed to identify an antibody response against the relevant neurotoxic amino acid or neurotoxic amino acid derivative. In one embodiment, the BMAA is conjugated to the immunogenic carrier protein to form an immunogenic BMAA carrier protein complex that is administered to a recipient animal and generates an immune response against the BMAA carrier protein complex; The product of the immune response is then collected and analyzed to identify an antibody response to BMAA or a BMAA derivative thereof. In one embodiment, BMAA is conjugated to KLH as a carrier protein to form a KLH-BMAA complex that is administered to a recipient animal and generates an immune response against the KLH-BMAA complex, Are analyzed to identify an antibody response to BMAA or a BMAA derivative thereof. In another embodiment, BMAA or a BMAA derivative is conjugated to BSA as a carrier protein to form a BSA-BMAA complex.

  As provided herein, in accordance with one aspect of the present invention, an antibody raised against an immunogenic hapten carrier conjugate may be a second antibody different from the carrier used in the immunogenic hapten carrier conjugate. Hapten conjugated to this carrier was subsequently tested. Furthermore, in accordance with this aspect, one skilled in the art can analyze the products of the immune response to the immunogenic hapten carrier complex in such a way that only the reactivity of the antibody binding to the hapten is detected. A second different carrier can be selected depending on the assumption that the recipient animal is less likely to be immunoreactive with the second carrier. In the following non-limiting exemplary examples, the KLH-BMAA conjugate is used to generate an immune response in rabbits, ie, an antiserum raised against the KLH-BMAA conjugate. After immunization, the antiserum is collected and the antiserum raised against the KLH-BMAA complex is evaluated using the BSA-BMAA complex to detect and characterize antibodies that bind to BMAA. As shown in the non-limiting exemplary embodiments in the Examples below, the BSA-BMAA conjugate can be used in an immunoassay to test antisera raised against the KLH-BMAA conjugate, For example, BSA-BMAA conjugates are used to coat microtiter plates for antibody capture assays and to test antisera to antibodies that react with BMAA. Without wishing to be bound by this theory, if KLH is not present during the testing of antisera, any antibody against the KLH portion of the immunogenic hapten carrier complex, if any, is present. Is also not detected. According to further aspects of the invention, and as illustrated in the non-limiting exemplary embodiments in the Examples below, the products of an immune response to immunization with an immunogenic KLH-BMAA complex (e.g., anti- Serum) can be immunoprecipitated with KLH to remove any anti-KLH antibodies present.

The conjugation of the hapten to the immunogenic carrier compound will depend on the type and number of reactive groups available on the hapten and carrier. Conjugation of the hapten to the immunogenic carrier compound can further include introducing a conjugate linker or spacer between the hapten and the carrier compound. Linkers and spacers minimize the structural conformational changes of the hapten during conjugation, and then promote presentation of the hapten to the immune system of the recipient animal for recognition by antibodies in an immunoassay. But can be selected by one skilled in the art according to various criteria, including but not limited to: Linkers and spacers include, but are not limited to, glutaraldehyde (Glu or GLU) and carbodiimide (Edc or EDC) linkers. In various embodiments, glutaraldehyde can be used to link the amino group to the amino group, and MBS (m-maleimidobenzoic acid-N-hydroxysuccinimide) can be used to link the amino group to the sulfhydryl group. Carbodiimides can be used to link amino groups to carboxyl groups. (See Antibodies: A Laboratory Manual, E. Harlow and D. Lane, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).
In one embodiment, BMAA or a BMAA derivative is conjugated to KLH as a carrier protein using glutaraldehyde (GLU) to form a KLH-GLU-BMAA (KGB) complex that is administered to a recipient animal, An immune response is generated against the complex, and the products of the immune response (antisera) are then analyzed to identify an antibody response with BMAA or its BMAA derivative. In one embodiment, the BMAA or BMAA derivative is conjugated to KLH as a carrier protein using carbodiimide (EDC) to form a KLH-EDC-BMAA (KEB) complex that is administered to the recipient animal, and the KEB complex , And the products of the immune response (antisera) are analyzed to identify the antibody response to BMAA.

  In one embodiment, the BMAA or BMAA derivative is conjugated to BSA as a carrier protein using glutaraldehyde (GLU) to form a BSA-GLU-BMAA complex that is administered to the recipient animal, and the BSA-GLU- An immune response is generated against the BMAA complex and the products of the immune response are analyzed to identify an antibody response with BMAA or a BMAA derivative thereof. In one embodiment, the BMAA or BMAA derivative is conjugated to BSA as a carrier protein using carbodiimide (EDC) to form a BSA-EDC-BMAA complex that is administered to the recipient animal, and BSA-EDC-BMAA An immune response is generated against the complex, and the products of the immune response are then analyzed to identify an antibody response with BMAA or its BMAA derivative.

  Non-limiting exemplary embodiments of the conjugation, immunization, and characterization procedures are disclosed in the Examples below. FIG. 1 shows a schematic of a non-limiting exemplary embodiment, wherein BMAA or a BMAA derivative is conjugated to KLH as a carrier protein using GLU to form a KLH-GLU-BMAA (KGB) complex. Or use EDC to form a KLH-EDC-BMAA (KEB) complex and administer each KLH-BMAA complex to a recipient animal to generate an immune response against the KLH-BMAA complex . BMAA or a BMAA derivative is also conjugated to BSA as a carrier protein using GLU to form a BSA-GLU-BMAA (BGB) complex or BSA-EDC-BMAA (BEB) complex using EDC. The body is formed and the BSA-BMAA complex is used to analyze the product of the immune response against the KLH-BMAA complex (antiserum) to identify the antibody response with BMAA or its BMAA derivative. .

  According to another aspect of the present invention, an immunogenic hapten carrier conjugate prepared using a defined crosslink (eg, GLU or ECD) as shown in the non-limiting exemplary embodiments found in the Examples below. Antibodies raised against the body were then tested against haptens conjugated to the same or different carriers via different crosslinks.

Antibody Selectivity for Neurotoxic Amino Acids and Neurotoxic Derivatives Antibody reactions with neurotoxic amino acids and their neurotoxic derivatives, combined with acceptable levels of selectivity, allow for acceptable high affinity and specificity for hapten targets Must have sex. Acceptable levels of selectivity include binding to the hapten target and having an acceptable low level of cross-reactivity with other antigens, particularly those that are structurally similar. Without wishing to be bound by this theory, useful antibodies (even from polyclonal sources), because the physicochemical properties of the target (epitope) exhibited by the hapten arise from a small number of nearly fixed residues. Even when antibodies from polyclonal and antibodies from different sources (eg, antibodies produced in different source animals) are compared, often a similar range of Has affinity. The affinity of the antibody for the hapten of interest can be determined using methods known in the art, such as an antibody capture assay, using different coating concentrations of the hapten, or increasing amounts of the hapten to samples where the baseline level of binding has been determined. Addition can be determined using a competitive inhibition assay for binding. Similarly, the selectivity of the antibody for the antibody of the hapten of interest can be determined using methods known in the art, such as competition assays, where other antigens are added to the sample where the baseline level of binding to the hapten has been determined. Can be determined.

  The product of an immune response, e.g., an antiserum or other antibody composition, targets multiple similar antigens, often binds with different affinities, and often targets different epitopes, Cross-reactivity is sometimes seen when containing antibodies. As mentioned above, hapten immunogens display very small, linear antigenic determinants, and anti-hapten antibodies generally have the same or overlapping targets (linear epitopes) to the extent that binding is mutually exclusive. Anti-hapten antibodies produced by the polyclonal method, very often have monoclonal properties, and sometimes have the properties of a single antibody, as long as the molecular selectivity is considered to be cross-reactive. Thus, a target or steric space can be formed by more than one different molecule. As described above, selectivity for the hapten of interest and cross-reactivity with other antigens can be determined for samples such as antisera or other antibody compositions using methods known in the art. it can.

  According to one aspect of the present invention there is provided an antiserum raised against a neurotoxic amino acid or a derivative thereof, wherein the antiserum is obtained without the serum being extensively purified or enriched and the method of the present invention. As used in the kit, it exhibits an acceptably high affinity and specificity for the hapten target in combination with an acceptably low level of cross-reactivity with other antigens. According to one aspect, antisera raised against BMAA carrier protein conjugates (anti-BMAA antisera) are combined with acceptable low levels of cross-reactivity with other antigens to achieve an acceptable high affinity for BMAA. Sexual and specific. According to this embodiment, the antiserum raised against the BMAA carrier protein conjugate (anti-BMAA antiserum) binds substantially to the BMAA conjugate and free BMAA, and does not substantially bind to structurally similar amino acids ( That is, it does not bind in appreciable amounts or in detectable amounts). According to this embodiment, the antiserum raised against the BMAA carrier protein complex (anti-BMAA antiserum) shows reactivity with the BMAA complex and with free BMAA, and the structurally similar amino acids are Even when present at very high concentrations, they do not substantially properly bind, detectably bind, or substantially cross-react with structurally similar amino acids. In a non-limiting exemplary embodiment disclosed in the Examples below, the antiserum raised against the BMAA carrier protein conjugate (anti-BMAA antiserum) is reactive with the BMAA conjugate and reacted with free BMAA. Alanine, glutamine, tyrosine, glycyl-glycine, glycine, leucine, phenylalanine, gamma-aminobutyric acid (GABA), glutamic acid, and sexually structurally similar amino acids, even when present at relatively high concentrations. Does not substantially cross-react with structurally similar amino acids, including aspartic acid. According to one aspect of the present invention, the anti-BMAA antisera provided herein can be used in the immunoassays and kits of the present invention without extensive purification or enrichment.

  According to another aspect, the purification step comprises removing undesired materials, such as non-specific antibodies or non-antibody proteins, prior to determining the relevant neurotoxic amino acid or neurotoxic amino acid derivative using the antiserum. Can be done. In accordance with one aspect of the present invention, the desired degree of specificity or purity is achieved by using methods known in the art to generate products of an immune response, such as antisera, raised against a neurotoxic amino acid or derivative thereof. Can be achieved by enrichment. Methods of purification and / or enrichment include, but are not limited to, protein A / G chromatography, ammonium sulfate precipitation, and affinity chromatography. In one embodiment, antisera raised against BMAA or a BMAA derivative conjugated to an immunogenic carrier protein is subjected to partial purification by ammonium sulfate precipitation. In a non-limiting exemplary embodiment shown in the examples below, antisera raised against BMAA or a BMAA derivative conjugated to KLH is immunoprecipitated with KLH to remove anti-KLH antibodies. Is subjected to partial purification. In one embodiment, antisera raised against BMAA or a BMAA derivative conjugated to an immunogenic carrier protein is applied to an affinity column having a BMAA or BMAA derivative linked to a column matrix.

  Immunoassays, antibodies, and kits can be used to distinguish isomers of the same compound, for example, to distinguish L and D forms of amino acids, or to distinguish non-neurotoxic isomers of a compound from neurotoxic isomers Provided to you. As shown in the non-limiting exemplary embodiments set forth in the Examples below, antibodies are provided that can distinguish between L-BMAA and D-BMAA.

  Methods for producing and using antibodies with high affinity for a target hapten and low cross-reactivity for similar haptens are known in the art. See, eg, Signature Immunology, Inc. Certain polyclonal antibodies, commercially available from (Salt Lake City UT), have high target specificity and low cross-reactivity to free (unbound) forms of certain amino acids. In one embodiment, an antibody is provided that has an acceptable high affinity for the target neurotoxic amino acid or neurotoxic amino acid derivative and has low cross-reactivity with other amino acids. In one embodiment, an antibody is provided that has an acceptable high affinity for BMAA or a BMAA derivative and has cross-reactivity with other amino acids.

  According to one embodiment, reacts with the free (unbound) form of the neurotoxic amino acid or derivative, or with the protein-bound form of the neurotoxic amino acid or derivative, or with both forms of the neurotoxic amino acid or derivative. , An antibody is provided. In one embodiment, an antiserum is provided that includes an antibody specific for the free form and an antibody specific for the protein-bound form. In one embodiment, an antibody is provided that is capable of recognizing both free and protein-bound forms. The protein-bound form of a neurotoxic amino acid or neurotoxic amino acid derivative includes a protein, eg, a protein-bound form of a neurotoxic amino acid or neurotoxic amino acid derivative incorporated into a polypeptide chain, and / or otherwise associated with the protein. Such as, but not limited to, protein binding of a neurotoxic amino acid or a neurotoxic amino acid derivative, which is attached to the protein by a covalent or non-covalent bond, or is conjugated to the protein through a spacer or linker group. I want to be understood.

  It is understood that compositions such as polyclonal antibodies, or antisera containing polyclonal antibodies, can include antibodies that recognize different epitopes. An antibody recognizing a free (unbound) form of BMAA or a BMAA derivative can be provided, an antibody recognizing a protein-bound form of BMAA or a BMAA derivative can be provided, and a free (non-bound) form of BMAA or a BMAA derivative and It should be understood that antibodies can be provided that recognize both protein-bound forms. Protein binding of a BMAA or BMAA derivative includes protein binding forms of the protein, eg, a BMAA or BMAA derivative incorporated into a polypeptide chain, and / or otherwise associated with the protein, eg, covalent or non-covalent binding It should be understood that these include, but are not limited to, protein binding of BMAA or a BMAA derivative, which is attached to the protein by a protein or conjugated to the protein through a spacer or linker group. As shown in the non-limiting exemplary embodiments in the Examples below, anti-BMAA polyclonal antibodies, or antisera containing antibodies raised against BMAA, comprise free BMAA, conjugated BMAA, and Reacts with protein-bound BMAA.

  In embodiments in which a monoclonal antibody is provided, one of skill in the art can individually identify an antibody-producing host or an antibody-producing host to identify those clones that have the desired level of stereospecificity for the neurotoxic amino acid or neurotoxic amino acid derivative. Clones can be screened.

  If deemed necessary, the affinity of the antibody for different haptens with similar configurations can be determined to determine the relative affinity for different targets, and for neurotoxic amino acids or neurotoxic amino acid derivatives. Can be mapped.

Immunoassays and Antibodies for Detecting the Presence of Neurotoxic Amino Acids Antibodies as provided herein are useful for detecting the presence, level (amount) of neurotoxic amino acids and neurotoxic amino acid derivatives in a sample, such as a tissue sample or an environmental sample. , And location, and can be used in immunoassays by those skilled in the art. The immunoassays of the present invention relate to a free (eg, unbound, unconjugated, cytoplasmic, circulating) form of a neurotoxic amino acid or a neurotoxic derivative thereof, a protein-bound form of a neurotoxic amino acid or a neurotoxic derivative thereof, or a neurological disorder. It can be performed to analyze complex forms (eg, glycoconjugates, lipid complexes, or carbamate adducts) of a neurotoxic amino acid or a neurotoxic derivative thereof, and any or all of these forms can be performed. Can be analyzed. One skilled in the art can determine which form of a neurotoxic amino acid or neurotoxic derivative thereof is present in a sample, and which form is a diagnostic or prognostic object for a given embodiment. . Antibodies and immunoassays as provided herein are used in conjunction with physicochemical methods to determine the presence, level, and location of the neurotoxic amino acids and neurotoxic amino acid derivatives described elsewhere in this disclosure. Can be done.

  Immunoassays and immunoassay formats suitable for use with the antibodies provided herein are known in the art. A homogeneous immunoassay format that does not require the separation of bound antibody-neurotoxic amino acid complexes from the rest of the assay components, as provided herein, the presence, levels, and levels of neurotoxic amino acids and neurotoxic amino acid derivatives, Suitable for determining placement. Homogeneous immunoassay formats requiring at least one step, often utilizing solid phase reagents such as magnetic particles or plastic beads to remove bound antibody-neurotoxic amino acid complexes from the remainder of the assay components Are suitable for determining the presence, level, and location of neurotoxic amino acids and neurotoxic amino acid derivatives, as provided herein. It should be understood that one skilled in the art can select and adapt the immunoassay format as needed. Suitable immunoassay formats include agglutination-based assays, precipitation-based assays ("Octalony" assays), radioimmunoassays, fluoroimmunoassays, chromogenic or colorimetric immunoassays, heterogeneous enzyme immunoassays, heterogeneous fluorescent immunoassays, fluorescence quenching or fluorescence enhancement. Sensitive homogeneous immunoassay, fluorescence polarization, enzyme-substrate-labeled immunoassay, prosthetic group-labeled immunoassay, enzyme modulator-labeled immunoassay (eg, using inhibitor labels), enzyme-labeled immunoassay, energy transfer immunoassay, chemically excited fluorescence Immunoassay, dual antibody steric hindrance immunoassay, or, for example, Harlow and Lane, Antibodies: A Laboratory Manual; Cold Spri ng Harbor Laboratory: other immunoassays as described in, but not limited to, Cold Spring Harbor, New York, 1988.

  Immunoassay formats used in immunoassays to detect neurotoxic amino acids and neurotoxic amino acid derivatives, particularly BMAA, include enzyme-linked immunosorbent assays (ELISA) using antibodies or antigens in an assayable detection system. However, the present invention is not limited to this, and suitable ELISA formats include, for example, Crowther, 1995. "ELISA. Theory and Practice" Methods Mol. Biol. 42: 1-223, Engval and Perlmann, 1971, "Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G" Immunochem. 8: 871-874, Davies, 1994, "Principles" in, The Immunoassay Handbook. D. Wild, ed. Stockton Press, New York, p. 3-47, and Harlow and Lane, Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, 1988, Appendix F. and Chalk. (1989, Appl Environ Microbiol 55: 1928-1933), and Metcalf et al. (2000, Water Research 34: 2761-2769) may include an antibody capture ELISA, a competition ELISA, or an indirect competition ELISA. The term "enzyme immunoassay" (EIA) is also commonly used to refer to the present immunoassay format.

  According to one aspect, an antibody as provided herein is an unlabeled antibody used as a "primary" antibody in a conventional immunoassay format. Thus, an antibody as provided herein can be detected and measured by a detectable secondary antibody that recognizes the primary antibody. Methods for selecting and using a detectable secondary antibody are known in the art. Suitable detectable secondary antibodies can be linked to enzymes such as horseradish peroxidase (HRP), alkaline phosphatase, lysozyme, glucose-6-phosphate dehydrogenase, and the like, and the coupling can be performed with various cross-linking agents, such as, for example, It can be achieved by conventional techniques using glutaraldehyde, dimaleimide or thiol reagents and the like, or linked to biotin or avidin, as described by Freytag et al. (1984, Clin. Chem. 30: 417-420). Or can be labeled directly, for example, with a radioactive label. Detecting the amount of secondary antibody bound to the primary antibody conjugated to the neurotoxic amino acid or neurotoxic amino acid derivative allows detection and quantification of the component.

  According to another aspect, the antibodies as provided herein can be used for direct detection and quantification of the binding of the antibody to a neurotoxic amino acid or derivative thereof according to protocols known in the art, e.g., radioactive. Labeled antibodies, or fluorescent moieties (fluorescent dye molecules), luminescent moieties, chemiluminescent moieties, colloidal gold, antibodies labeled with dye moieties, enzyme-linked antibodies, biotin-labeled antibodies, avidin-labeled antibodies, streptavidin-labeled antibodies, or other The labeled antibody can be directly labeled with a detectable moiety. In a non-limiting exemplary embodiment, the labeled anti-BMAA antibody provides, for example, a biotin-labeled anti-BMAA antibody.

  According to one embodiment, an antibody capture immunoassay is provided to determine the presence and affinity of an antibody as provided herein, ie, the antibody response with a neurotoxic amino acid or neurotoxic amino acid derivative. In this assay, a known amount of unlabeled neurotoxic amino acid or derivative is, for example, used in a multiwell microtiter plate using serially diluted stock solutions such that each well contains a known amount of neurotoxic amino acid or derivative. An antibody as provided herein (the "primary" antibody) is added to an assay well by coating a series of wells of a neurotoxic amino acid attached to a solid substrate. Alternatively, by binding to a derivative, it is "captured" on a solid support and detected by a detectable secondary antibody that recognizes the primary antibody. In each assay, the amount of primary antibody that is bound ("captured by") to the neurotoxic amino acid or derivative bound to the solid support is bound to the primary antibody by methods known in the art. It is determined by measuring the amount of a secondary antibody that can be detected. In one embodiment, antisera raised against a neurotoxic amino acid or neurotoxic amino acid derivative is used in an antibody capture assay to determine the level of antibody response with the neurotoxic amino acid or neurotoxic amino acid derivative present in the antiserum. Is detected. In one embodiment, antisera raised against BMAA or a BMAA derivative is used in an antibody capture assay to measure antibody binding to the BMAA or BMAA derivative linked to a solid support.

  According to one aspect, to determine the ability of an antibody as provided herein to bind to a free (unbound) form of a neurotoxic amino acid or a neurotoxic amino acid derivative used to generate the antibody. , An indirect competitive ELISA is provided. In this assay, a known amount of unlabeled neurotoxic amino acid or derivative is prepared, for example, using a multi-well microtiter plate using serially diluted stock solutions such that each well contains a known amount of neurotoxic amino acid or derivative. To the solid support by coating a series of wells. An antibody as provided herein is used as a "primary" antibody, and free neurotoxic amino acid or neurotoxic amino acid derivative is added to an assay well and a known amount of free neurotoxic amino acid or neurotoxic amino acid derivative is added. Antibody capture in the presence is compared to antibody capture in the absence of free neurotoxic amino acids or neurotoxic amino acid derivatives. As disclosed in the non-limiting exemplary embodiments set forth in the Examples below, an indirect competitive ELISA was performed to determine the reactivity of antisera produced against the BMAA complex with free BMAA and provided. Antibodies that bind to free BMAA can be constructed.

  According to one aspect, an immunoblot assay is provided for determining the ability of an antibody as provided herein to bind a neurotoxic amino acid or a neurotoxic amino acid derivative in a sample. According to one aspect, the antibodies as provided herein can be used on whole cell extracts or protein preparations from a sample to determine if there is a reaction with any of the components present in the sample. Immunoblot assays can be performed using the "dot blot" format. According to another embodiment, the immunoblot assay can be performed in a "Western blot" format, wherein the proteins in the protein-containing extract of the sample separate proteins based on, for example, size and / or loading The separated proteins are then transferred to a cell membrane, such as nylon or nitrocellulose, and the antibodies as provided herein are used in an immunoassay, The protein-binding neurotoxic amino acid or neurotoxic amino acid derivative is detected above. In one embodiment, using antibodies raised against BMAA (BMAA-KLH complex) as provided herein, BMAA or BMAA by Western blot of a protein preparation from a tissue or environmental sample. The BMAA derivative is detected.

  According to one aspect, a protein extract is prepared from a tissue or environmental sample, and an immunoassay for BMAA or a BMAA derivative is performed on the protein extract as provided herein to recognize protein binding by the antibody. Should be understood to indicate that the sample may contain protein-bound BMAA or a BMAA derivative. In one embodiment, a tissue sample is obtained from a subject and the tissue sample is contacted with an antibody composition comprising at least one antibody that binds BMAA under conditions that allow binding of the antibody to BMAA. By detecting antibodies that bind to BMAA in the subject, the subject is screened for exposure to an environmental source of BMAA, and antibody binding to a tissue sample indicates that the tissue contains BMAA; Indicates that the subject has been exposed to an environmental source of BMAA.

  In one embodiment, an environmental sample is obtained and contacted with an antibody composition comprising at least one antibody that binds BMAA under conditions that allow binding of the antibody to BMAA, wherein the BMAA in the environmental sample is contacted. An environmental source of BMAA is detected by detecting the antibody bound to, and antibody binding to the environmental sample indicates that the environmental sample contains BMAA and, thus, is an environmental source of BMAA. In one embodiment, the environmental sample is contacted with an antibody composition comprising at least one antibody that binds BMAA under conditions that allow binding of the antibody to BMAA, and the antibody bound to BMAA in the environmental sample is detected. And comparing antibody binding to the environmental sample with antibody binding to the sample of cyanobacteria to determine whether antibody binding to the environmental sample indicates the presence of a cyanobacterial source of BMAA. A cyanobacterial source of BMAA is detected.

  In one embodiment, an immunoblot ("Western Blot") assay of a protein extract of a tissue sample comprises an antibody composition comprising at least one antibody that binds BMAA (eg, an antiserum raised against BMAA). Performed to identify the protein bands recognized by the antibody. In one embodiment, an immunoblot or “Western blot” assay of a protein extract of an environmental sample uses an antibody composition comprising at least one antibody that binds to BMAA (eg, an antiserum raised against BMAA). To identify the protein band recognized by the antibody. In one embodiment, an immunoblot or "Western blot" assay of a protein extract of a keratinous tissue sample is performed to identify the protein bands recognized by the antibodies of the invention. In another embodiment, an immunoblot or "Western blot" assay of a protein extract of a neurotoxic tissue sample is performed to identify the protein bands recognized by the antibodies of the invention.

  According to one aspect, antibodies that have a reaction with protein-bound BMAA are used for in situ labeling or imaging techniques, such as immunohistochemical techniques, wherein the antibody is a protein-binding neurotoxic amino acid or a neurotoxic amino acid derivative. Binds to containing lesions. Protocols for immunocytochemistry for detecting small molecules such as amino acids in histological specimens are described, for example, in Signature Immunology, Inc. (Salt Lake City UT) rabbit polyclonal antibody, for example, http: // www. immunologics. com / hpi. html. Is well known in the art of high performance immunocytochemistry on epoxy resin embedded specimens using gold-conjugated or fluorophore-conjugated anti-rabbit secondary antibodies, as described in US Pat.

  Amplification can be used to enhance signal strength and / or selectivity. Similarly, one skilled in the art can modify neurotoxic amino acids and neurotoxic amino acid derivatives to obtain labeled conjugates detectable by the immunoassays of the present invention.

  One skilled in the art can use immunoassays and antibodies as provided herein to analyze samples for neurotoxic amino acids and neurotoxic derivatives thereof, including tissue samples and environmental samples. But not limited thereto. Tissue samples obtained from living subjects (live in vitro, in vitro), tissue samples present in living subjects (in vivo), stored using immunoassays as provided herein. Archived samples, such as tissue, biopsy and / or necropsy samples, or museum samples can be analyzed. The preserved tissue can be frozen tissue, a histological specimen, tissue dried on a solid storage medium, or other form of preserved tissue.

Antibodies as provided herein can be used in vivo in imaging and diagnostic applications to detect neurotoxic amino acids and neurotoxic derivatives thereof in a subject. In particular, antibodies as provided herein can be used for in vivo diagnostic imaging to detect neurotoxic amino acids or neurotoxic derivatives thereof in body fluids, or in body lumens, or in other body tissues. it can. In one embodiment, the antibody reaction with the protein-bound BMAA is introduced into a body lumen, such as the spinal cord, blood vessels, ureter, urethra, esophagus, cervix, uterus, or bladder, and the antibody is injected into the body fluid in the lumen Of BMAA, or BMAA-containing proteins in tissues on the wall in the lumen. In another embodiment, an antibody reaction with a protein-bound BMAA is introduced into a tissue or organ, for example, by perfusion, and the antibody can bind to a BMAA-containing protein in situ in the tissue or organ. The antibodies of the present invention are given in a diagnostically effective dose that allows the detection of protein-bound BMAA for a particular application. For in vivo imaging, the antibody can be labeled or otherwise linked to a detectable marker so that the antibody can be directly detected. In another embodiment, a detector, such as a secondary antibody, is introduced into the body lumen and detects the antibody that is bound to BMAA. Detectable markers that can be linked to the antibodies of the invention include radioisotopes such as ¹³¹ I, ¹²⁵ I, ¹²³ I, ¹⁸ F, ¹⁹ F, ¹¹ C, ¹³ C, ¹⁴ C, ⁷⁵ Br, ⁷⁶ Br, or H. Contains elements. The marker can be a paramagnetic compound, for example, a compound that includes a lanthanide. The marker may be a contrast agent suitable for detection by contrast-enhanced ultrasound, for example, a microbubble having a suitable biocompatible shell conjugated to an antibody and a heavy gas (perfluorocarbon or nitrogen). For in vivo diagnostic imaging, the type of detection device that can be used is a major factor in selecting a detectable marker, so the marker can be X-ray (e.g., ¹²⁵ I, ⁵⁷ Co, technetium-99m ( ^99m Tc)), ultrasonic waves (eg, perflutoren ultrafine bubbles), MRI (eg, gadolinium, ¹⁹ F, ¹ H), PET ( ¹⁸ F), computed tomography (CAT), magnetic spectroscopy (MRS), It can be selected for photon emission computed tomography (SPECT), bioluminescence imaging (BLI), or other applications. In some embodiments, the labeled antibody bound to BMAA is detected or measured locally in an organ or tissue. In some embodiments, the labeled antibody that is bound to BMAA is systemically scanned by scanning all or a portion of the subject using imaging techniques such as X-ray, ultrasound, PET, or MRI. Measured. Pharmaceutical compositions suitable for administration for in vivo imaging and diagnostic applications are provided, wherein the pharmaceutical composition comprises a detectable antibody that allows for an antibody reaction with protein-bound BMAA and detection of an antibody bound to BMAA. Markers are included. Protocols for introducing and detecting markers, such as antibodies, for detecting lesions are found in US Patent Nos. 5,716,595, 6,375,925, and 6,782,289. And incorporated herein by reference.

Sample Preparation The immunoassays, antibodies, and kits of the present invention can be used in preparation steps to adapt certain properties of a sample, for example, to prepare a tissue sample for analysis, or to provide a subject for in vivo measurements / images. It should be understood that the method can include a step for preparing an environmental sample, or a step for preparing an environmental sample for analysis. Sample preparation can include, but is not limited to, mechanical or chemical disruption of the sample, including, but not limited to, from a sample for detection by immunoassays and antibodies as provided herein. Release of BMAA includes, but is not limited to, hydrolysis (eg, acid hydrolysis), enzymatic digestion, or solvent extraction (solvent resolution).

  In one embodiment, keratinous tissue, such as hair, is hydrolyzed using a strong acid and heat, and the BMAA immunoassay is performed on the neutralized hydrolyzate. In another embodiment, the hair is enzymatically digested and immunoassayed for BMAA using a protease mixture containing a reducing agent and a detergent, for example, as described in US Pat. No. 6,949,344. Is performed in digestion. In another embodiment, neural tissue, such as brain tissue, is first homogenized under acidic conditions (eg, 0.1 N trichloroacetic acid) and centrifuged to release free amino acids and precipitate proteins. The precipitate is then subjected to hydrolysis using a strong acid and heat (eg, 110 ° C. for 24 hours, 6N HCl), followed by immunoassay of the neutralized supernatant to determine free BMAA in the sample. Once determined, an immunoassay of the hydrolyzate of the neutralized precipitate is performed to determine the protein-bound BMAA released from the precipitate by hydrolysis.

  In one embodiment, an environmental sample comprising cellulose is treated with cellulase to release cell contents and cell wall material for determining BMAA. In one embodiment, an environmental sample comprising chitin is treated with chitinase to release cell contents and cell wall material for determining BMAA.

  In one embodiment, the sample is processed to obtain a plurality of sample fractions and the immunoassay of the invention is used to determine BMAA in one or more of the resulting sample fractions. In one embodiment, the sample can be processed to obtain a protein fraction and a soluble fraction, for example, as disclosed in US Pat. No. 7,256,002, where cyanobacteria, Cycad species Tissue, fruit bat hair and skin, human brain tissue samples are processed to remove free amino acids (from soluble or cytoplasmic fractions) to obtain a protein fraction that is supposed to contain protein-bound BMAA. The protein fraction was then hydrolyzed and the BMAA in the hydrolyzate was determined using HPLC. In another embodiment, the sample can be extracted with different ambipolar solvents to obtain, for example, aqueous and lipophilic fractions.

  According to one aspect, the sensitivity of the BMAA immunoassay increases the concentration of BMAA in the sample to a level suitable for current immunoassay procedures, and removes potentially interfering substances, prior to immunoassay, sample concentration and / or sample clarification. Can be enhanced. In a non-limiting exemplary embodiment, commercially available solid phase extraction (SPE) sorbents were evaluated for their ability to retain BMAA from solution and then release BMAA in the elution step. Based on the results from the initial SPE screening, further testing of different polymeric SPE phases suggests that strong cationic polymeric SPE sorbents (eg, StrataXC) have acceptable BMAA retention and elution properties It was shown to be possible. In one embodiment, the sample is subjected to a preclean using an SPE sorbent prior to immunoassay of the sample, as provided herein. In one embodiment, multiple SPE phases, as provided herein, are used for a multifaceted approach to BMAA retention and clarification from a sample using continuous SPE extraction of the sample prior to immunoassay.

The present invention screens at least one tissue sample from a subject to detect for the presence of BMAA to screen for a subject having or at risk of having a neurological disorder. Immunoassays, antibodies, and kits to perform As provided herein, neurological disorders (also known as neurological disorders or diseases or neurological disorders) include the central nervous system (brain, brainstem, and cerebellum), the peripheral nervous system. (Including cranial nerves), and the autonomic nervous system, some of which are located in both the central and peripheral nervous systems. It is understood that a neuropathy can have a complex etiology, such that one or more environmental or genetic factors can contribute to the development of the neuropathy in a subject. Neuropathy includes well-characterized disorders or syndromes, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), or Parkinson's disease, or signs observed in multiple disorders (eg, aphasia) ) Or symptoms (eg, tremor). It is further understood that the development of a neurological disorder in a subject can be due to one factor, or a combination of factors. Similarly, it should be understood that a particular neuropathy in a subject may be due to different factors, or different combinations of factors, that produce the same neuropathy in other subjects. Immunoassays as provided herein are suitable for screening for neurological disorders in which one or more environmental or genetic factors play a role.

  Screening methods include a method for diagnosing one or more neuropathies in a subject, a method for confirming the diagnosis of one or more neuropathies in a subject, and developing one or more neuropathies in a subject. For predicting the risk or likelihood of developing a subject, a method for predicting the severity of a neuropathy in a subject, and exposing the subject to a neurotoxic amino acid or neurotoxic derivative thereof associated with the development of a neuropathy , But is not limited thereto. The method of the present invention may be performed in a repetitive manner to determine the presence and level of a neurotoxic amino acid or a neurotoxic derivative thereof in a subject, and / or a time sequence for the presence and level of a neurotoxic amino acid or a neurotoxic derivative thereof in an environmental sample. Includes a method for generating data. The method includes correlating the presence of a neurotoxic amino acid or a neurotoxic derivative thereof in a tissue sample from the subject with other physical or psychological decisions regarding the neurological disorder being assessed. The method further comprises correlating the level of the neurotoxic amino acid or neurotoxic derivative thereof in one or more tissue samples from the subject with other physical or psychological decisions regarding the neurological disorder being evaluated. Including. In one embodiment, a tissue sample is obtained from a subject diagnosed as suffering from a neuropathy, BMAA levels are determined, and these are included as part of a method for diagnosing one or more neuropathies. The result is compared to other physical or psychological measurements of the subject.

  Screening using the immunoassays, antibodies, and kits of the present invention may result in the risk of having or developing one or more neuropathies to refine or confirm the diagnosis of one or more neuropathies. Or to rule out other possible diagnoses. In one embodiment, an immunoassay of the invention is performed to detect the presence of BMAA in a tissue sample from a subject who is not currently presenting symptoms to one or more neuropathies. In another embodiment, an immunoassay of the invention is performed to detect BMAA levels in a tissue sample from a subject that is currently symptomatic for one or more neuropathies. In another embodiment, a method for performing an immunoassay of the invention to detect the presence of BMAA in a tissue sample from a subject suspected of having a neuropathy, and to diagnose one or more neuropathies As a part of these, these results are compared to other physical or psychological measures of the subject. In a further embodiment, the immunoassay of the invention is performed in an iterative manner, measuring BMAA levels in the tissue sample over time and determining whether the subject may be at risk of developing a neurological disorder and may require additional monitoring. Identify the body. In a further embodiment, the immunoassays of the invention are performed to detect the presence of BMAA in one or more tissue samples, and those skilled in the art will be able to determine the level of BMAA physically or psychologically with respect to the neurological disorder being assessed. Correlate with other measures, such as determination, and / or genetic analysis of the subject (eg, family history and / or genotype tissue sample), the risk or likelihood of having or developing a neurological disease Can be determined.

  According to another aspect, ingesting tissue samples at repeated intervals over an extended period of time, performing an immunoassay to detect the presence of BMAA in each tissue sample, and providing time-continuous data on BMAA levels useful for longitudinal studies This provides a method for longitudinal studies of neuropathy. In yet another embodiment, the immunoassays of the invention are performed repeatedly to detect the presence of BMAA in a tissue sample from a subject over an extended period, wherein the level or amount of BMAA in each sample is determined over time. Together provide data on BMAA accumulation in tissues, which is useful in predicting the likelihood and / or timing and / or severity of future onset of one or more neurological disorders. In yet another embodiment, the immunoassays of the invention are performed repeatedly to detect the presence of BMAA in a tissue sample from a subject over an extended period, wherein the level or amount of BMAA in each sample is determined over time. Along with providing data on BMAA release from tissues, which is useful in predicting the likelihood and / or timing and / or severity of one or more neurological disorders in the future.

  The present invention relates to Parkinson's disease (PD), Alzheimer's disease (AD), progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), and ALS-PDC (Guarum ALS-PDC or lytico-bodydig). Immunoassays, antibodies, and kits for use in screening for neuropathy are provided, including, but not limited to, neuropathological diseases known as disease. The teachings of the present disclosure provide sufficient guidance to identify other neurological disorders for which the present invention provides screening methods, and those skilled in the art will be able to practice the methods of the present invention and to obtain Detect the presence of BMAA, determine the level of BMAA, and then compare these levels with other signs of neurological disease in the subject, and determine the level of BMAA in the sample between the level of BMA and the sign of the particular neurological disorder. It can be determined whether a correlation exists. Because different diseases often share similar signs and symptoms (eg, tremor, dementia, aphasia), the immunoassays, antibodies, and kits of the invention are used as part of an initial screen for neurological disease. It may only be suitable where the results of the immunoassay-based initial screen are trusted to determine which further tests are needed for a full evaluation. For example, a subject with ALS-PDC may have symptoms similar to Alzheimer's disease or Parkinson's disease, or both, and ALS-PDC is considered to be a separate disease, but ALS-PDC The subject may also have Alzheimer's disease or Parkinson's disease. Similarly, subjects with Alzheimer's disease and subjects with other forms of dementia may have some similar symptoms, but may differ in the BMAA content of various tissues. Thus, measurement of BMAA levels in a subject can help identify which neuropathy is present and contributes to the signs and symptoms observed in the subject.

  According to one embodiment, any of the tissue samples from the subject can be used to perform the immunoassay of the invention. In one embodiment, the tissue sample is analyzed to detect the presence of BMAA. In another embodiment, detecting the presence of BMAA comprises determining the amount of BMAA present in the tissue sample. In another embodiment, a tissue sample can be analyzed to detect the location of BMAA in an in vivo or in vitro tissue as well as the location of BMAA. In another embodiment, the tissue sample is processed to obtain at least two sample fractions, and at least one fraction is analyzed to detect the presence of BMAA. The level (amount) of free and / or protein-bound BMAA can be determined (quantified) according to the nature of the tissue sample and the questions to be answered in a particular embodiment. In some embodiments, it may be desirable to determine the levels of both free and protein-bound BMAA. In other embodiments, it may be desirable to determine only free BMAA levels. In other embodiments, it may be desirable to determine only protein-bound BMAA levels. In some embodiments, the tissue is analyzed such that both free and protein-bound BMAA are recovered in a single sample fraction (hydrolysate) that is analyzed to determine total BMAA levels in the sample. Can be completely chemically separated (eg, by hydrolysis).

  The tissue sample can be obtained from a living subject, present in a living subject, or a stored specimen, such as a stored tissue, biopsy and / or necropsy sample, or a museum sample. It may be obtained from a specimen. The preserved tissue can be frozen tissue, a histological specimen, tissue dried on a solid storage medium, or other form of preserved tissue. Suitable tissue samples include, but are not limited to, neural or non-neural tissue. Neural tissue may be associated with the central nervous system (CNS), including brain tissue or cerebrospinal fluid (CSF), or with the peripheral nervous system (PNS). Neural tissue can include tissue present in a living subject, including but not limited to cerebrospinal fluid (CSF) suitable for in vivo imaging. The non-neural tissue can be keratinous tissue or non-keratinous tissue, including, but not limited to, blood, serum, saliva, or urine. Non-neural tissue can be analyzed in vitro or in vivo. For example, in vitro analysis of blood can involve removing blood from a subject and analyzing a blood sample, while in vivo analysis of blood involves detecting and imaging blood in a body lumen, such as a blood vessel. Can be accompanied.

  Keratinous tissue includes, but is not limited to, hair, skin, nails, including fingernails or toenails, wings, claws, hooves, or horns. In accordance with one aspect of the present invention, a sample of keratinous tissue from a subject collected at multiple time points, eg, a hair or skin sample, is analyzed to detect the presence of BMAA and, optionally, to determine BMAA levels. can do. In one embodiment, the hair is analyzed to detect the presence of BMAA. In one embodiment, the hair is analyzed to detect the total level (amount) of BMAA in the sample. In one embodiment, the hair may be analyzed to detect free (eg, in separate sample fractions) free and protein-bound BMAA and to determine the level (amount) of free and protein-bound BMAA. . In another embodiment, the hair is analyzed to detect only free BMAA. In another embodiment, the hair is analyzed to detect only protein-bound BMAA. In another embodiment, the skin is analyzed to detect BMAA. In one embodiment, the skin is analyzed to detect the total level (amount) of BMAA in the sample. In one embodiment, the skin may be analyzed to detect free and protein-bound BMAA separately (eg, in separate sample fractions) and to determine free and protein-bound BMAA levels. In another embodiment, the skin is analyzed to detect only free BMAA levels. In another embodiment, the skin is analyzed to detect only protein-bound BMAA levels.

  In another embodiment, the brain tissue may be analyzed to detect the presence of BMAA, and the brain tissue may be analyzed to determine the level of BMAA in the tissue. In another embodiment, a sample of spinal fluid (CSF) may be analyzed in vivo or in vitro to detect the presence of BMAA, and the CSF may be analyzed to determine BMAA levels in the fluid. Brain or CSF tissue can be analyzed to determine the levels of protein-bound BMAA, free BMAA, or both protein-bound and free BMAA, and the protein-bound BMAA can be bound to neural proteins or other proteins.

Screening for Environmental Factors Associated with Neuropathy According to one aspect, immunoassays, antibodies, and kits are provided for screening for environmental factors associated with neuropathy by detecting the presence of BMAA in an environmental sample. You. Screening as provided herein includes testing environmental samples to determine the actual or potential exposure of a subject to a neurotoxic amino acid or neurotoxic derivative thereof associated with a neurological disorder. But not limited to this. Environmental samples can be obtained from ingested substances, such as water samples or food samples. The environmental sample can be, for example, a substance that is intentionally consumed, such as drinking water, or a plant or animal that is part of the food supply or food chain. Alternatively, the environmental sample may be a substance that is inadvertently ingested (eg, an organism whose contents or secretions become related to other ingested substances (eg, cyanobacterial symbionts present in edible plants). Or substances from cyanobacteria in water for washing or drinking).

  In one embodiment, the immunoassays, antibodies, and kits of the present invention are used to determine the actual or potential exposure of a subject to BMAA to determine (quantify) BMAA levels in an environmental sample. Provided. Measurement of BMAA levels in environmental samples leads to a determination of potential or actual exposure to BMAA, and these measurements indicate the potential for developing neurological disorders in subjects exposed to these environmental samples. Can be used to predict As disclosed in US Pat. No. 7,256,002, HPLC analysis of samples from cyanobacterial archives showed that almost all strains tested produced BMAA. In addition, as disclosed in U.S. Patent No. 7,256,002, BMAA found in cycad tissue is thought to be produced by cyanobacterial symbionts that are incorporated into cycads, thus rendering humans and fruit bats ( Other organisms that feed on cycads, such as "flying fox" (bats), are thought to consume BMAA of cyanobacterial origin.

  According to another embodiment, the environmental sample is water, which is known to contain cyanobacteria. In another embodiment, the environmental sample is water suspected of containing cyanobacteria. In another embodiment, the environmental sample is water whose contents are unknown. In another embodiment, the environmental sample can be a food animal, such as a fish, bird, deer, or domestic animal that consumes the water contained by cyanobacteria. In another embodiment, the environmental sample can be a lichen, or moss, or a moss, that contains or lives in symbiosis with cyanobacteria.

  In another embodiment, the environmental sample can be a marine or freshwater algae, or a marine or freshwater fungus that contains or lives in symbiosis with cyanobacteria. In another embodiment, the environmental sample can be a marine or freshwater invertebrate that contains or lives in symbiosis with cyanobacteria. In another embodiment, the environmental sample can be stromatolite, petrochemical deposit, or mineral deposit left by cyanobacteria. In another embodiment, the environmental sample is a plant, lichen, moss, algae, marine invertebrate that contains cyanobacteria or stromatolites, petrochemical deposits, or mineral deposits left by the cyanobacteria. Food animals, such as reindeer, caribou, deer, mousse, saltwater or freshwater fish, birds, reptiles, or domestic animals.

  In accordance with another aspect, an environmental sample determines whether a sample is associated with a neurological disorder by detecting the presence of a neurotoxic amino acid, particularly a cyanobacterium that produces BMAA, in the environmental sample. Screened to determine. The immunoassays, antibodies, and kits of the invention are performed to detect the presence of the genus Cyanobacteria, including, but not limited to, Nostoc and Anabena. By screening environmental samples to detect cyanobacteria that produce BMAA, it is possible to determine the actual or potential exposure of a subject to environmental factors associated with a neurological disorder.

  According to another aspect, a plurality of environmental samples are tested to determine the presence and levels of neurotoxic amino acids, particularly BMAA, that are associated with neurological disorders at different levels throughout the food chain. Without wishing to be bound by this theory, bioconcentration of factors associated with neuropathy, for example, BMAA, may result in higher consumption of organisms from higher trophic levels than consumption of organisms from lower trophic levels. Can result from the accumulation of factors in biological tissues at different trophic levels, which can result in higher exposure to neurotoxicity. In one exemplary embodiment, a plurality of environmental samples are tested in the food chain, including those known to eat cycad coral roots, cycad leaves, cycad seeds, and cycad seeds. Tissue samples from fruit bats (bats) are included. In another embodiment, a plurality of environmental samples are tested in the food chain, including water, aquatic plants, and edible animals that consume the water and aquatic plants, such as fish, birds, wild animals or domestic animals. And carnivores that feed on herbivores. In one embodiment, multiple environmental samples may be tested to determine if a factor such as BMAA is found in a particular food chain. After testing multiple environmental samples, the levels of neurotoxic amino acids, eg, BMAA, can be compared and analyzed for evidence of accumulation or bioconcentration in the food chain.

  According to a further embodiment, in addition to testing the environmental sample for neurotoxic amino acids associated with a neurological disorder, a tissue sample from the subject is also analyzed. Screening at least one tissue sample from a subject to determine the accumulation or bioconcentration of environmental factors (neurotoxic amino acids, particularly BMAA) in the food chain and to provide useful data, Useful for correlating the levels of these environmental factors (eg, BMAA) at each stage of the disease with the frequency or severity of neurological disorders in subjects consuming substances from various trophic stages of the food chain I will provide a. In one embodiment, a tissue sample from a subject with a symptom or diagnosis of a neurological disorder is analyzed to detect neurotoxic amino acids associated with a neurological disorder, particularly BMAA. In another embodiment, a tissue sample from a subject who is asymptomatic for a neurological disorder is analyzed to detect neurotoxic amino acids associated with a neurological disorder, particularly BMAA. This aspect of the invention provides a powerful means for linking neuropathy to exposure to environmental factors known or suspected to be associated with neuropathy.

  In a non-limiting exemplary embodiment, in the disclosed U.S. Patent No. 7,256,002, an increase in BMAA levels is caused by a known increase in the level of BMAA to known or suspected food sources. Following exposure, it was detected in the brain tissue of subjects who died of ALS-PDC, i.e., subjects who died of ALS-PDC were Chamorro who had eaten a traditional chamorro diet several times in their lives. Yes, this could have included cycad flour, and could include fruit bats (bats), and measurements of BMAA levels in a fruit bat sample showed high levels of BMAA Leading the prediction that fruit bat consumption can result in a BMAA dose equivalent to the dose obtained by eating 174 kg to 1,014 kg of processed cycad flour . In addition, elevated levels of BMAA are detected in one Chamorro subject who is asymptomatic of ALD-PDC and has died from other causes, which has developed (ALD-PDC) Chamorro and Consistent with the findings of neurofibrillary tangles in both brain tissues of unaffected (asymptomatic) Chamorro. In contrast, another Chamorro who was asymptomatic for ALS-PDC and died of other causes, had no detectable BMAA levels in brain tissue.

  Another aspect of the present invention provides a method for detecting environmental pollution by an environmental factor associated with a neurological disorder. In a non-limiting exemplary embodiment, in the disclosed US Patent No. 7,256,002, elevated levels of BMAA are detected in brain tissue of a non-Chamoro (Canadian) subject suffering from Alzheimer's disease. And non-Chamoro (Canadian) with progressive supranuclear nucleus (PSP), where these subjects were exposed to environmental sources of BMAA several times in their lives. It was shown that. According to another aspect, bioconcentration of the cyanobacterium BMAA can occur through the food chain, which results in accumulation in the tissues of the subject. Since the frequency of disease in the population exposed to neurotoxicity is a function of dose, even low levels of progressive neuropathy are associated with exposure to low levels of BMAA in water supply contaminated with cyanobacteria. May be. Thus, environmental screening as provided herein can be performed to investigate possible environmental sources of BMAA or other environmental factors associated with neuropathy. Environmental screening as provided herein prevents or minimizes the exposure of another subject to BMAA or other environmental factors associated with a neurological disorder, and thereby, a neurological disorder associated with BMAA or other factors. Can be performed to reduce the risk of developing

  According to a further aspect, the immunoassays, antibodies, and kits of the invention protect a subject from exposure to environmental factors associated with a neurological disorder by screening an environmental sample prior to the subject eating. Can be used for In one embodiment, immunoassays, antibodies, and kits are provided for testing food samples containing botanicals or animals for BMAA. In another embodiment, immunoassays, antibodies, and kits are provided for testing water supply for BMAA. Kits for environmental screening for BMAA include materials for performing the methods of the present invention for testing water supplies, food sources, and other environmental samples to protect subjects from exposure to BMAA. including. According to another aspect, the immunoassays, antibodies, and kits of the invention can be used for public health purposes, for example, to indicate contamination of a water supply or food source by cyanobacteria producing BMAA.

Kits for Screening for Neurotoxic Amino Acids The present invention provides kits that include means for performing the immunoassays of the present invention. In one embodiment, the present invention provides a kit for screening a subject having or at risk of having a neurological disorder, the kit comprising the presence of BMAA in a tissue sample from the subject. Immunoassay to determine In another embodiment, the present invention provides a kit for screening an environmental sample for an environmental factor associated with a neurological disorder by determining the presence of BMAA in the sample, wherein the kit comprises: Includes an immunoassay to determine the presence of BMAA. A kit of the invention can include a means for analyzing a plurality of types of samples, for example, the kit provides a means for analyzing a tissue sample from a subject, as well as an environmental sample such as a water or food sample. May be included. Alternatively, a kit of the invention may include only means for analyzing one or several types of samples, for example, a kit may include only means for analyzing a keratinous tissue sample, such as hair.

  Such a kit may include a carrier means compartmented to receive one or more container means, such as vials, tubes, etc., each of the container means being one of the separate components used in the method. Including one. For example, one of the container means may include an antibody that binds to BMAA, and the components may be in liquid or lyophilized form, as desired. The kit may include additional container means, including separate components for detecting antibodies that bind to BMAA. If the antibody that binds to BMAA is detectably labeled, the kit may include one or more additional container means containing the reagents required to detect the labeled antibody that binds to BMAA; , Any container means need to perform the detection reaction. For example, if the antibody that binds to BMAA is labeled with biotin, the container means may include an avidin or streptavidin reporter molecule and a reagent capable of producing biotin-avidin or biotin-streptavidin, while another The container means may include reagents for removing unbound antibody and reporter molecules. If the antibody that binds to BMAA is not detectably labeled, the kit may further comprise a separate element for detecting the antibody that binds to BMAA using a detectably labeled secondary antibody. Which include the reagents necessary to detect secondary antibody binding to antibodies that bind to BMAA. For example, the kit may include additional container means containing a horseradish peroxidase (HRP) labeled secondary antibody, in liquid or lyophilized form, as desired. Another container means contains reagents for incubating the secondary antibody with the sample and the antibody that binds to BMAA, while another container means contains reagents for removing unbound antibody after incubation. . Another container means contains reagents for detecting HRP activity, eg, HRP substrate. If necessary, further container means include means for visualizing the HRP product. Depending on the immunoassay format, the sample can be planarized into container means for a particular step, or the contents of the container can be removed for use.

  Preferably, the kits of the present invention, as provided herein, include all components and reagents required to perform an immunoassay, such as containers for manipulating samples and containers for performing reactions. , As well as reagents for introducing a reaction that is observable or otherwise measurable to determine BMAA in the sample. Thus, the kit may include a vehicle that is compartmentalized in a receiving container means that contains all the components for which the kit is provided.

  A kit of the invention may also include a "control" antibody, eg, a null serum or antibody that does not bind to BMAA. Kits of the invention may include a "positive control" sample, which is known to contain BMAA. Kits of the invention may include a "negative control" sample, which is known not to contain BMAA. Kits of the invention may include a panel of "control" or "standard" samples of known amounts of BMAA, whereby a standard curve may be constructed for quantification and calibration. The kit may include means for analyzing a plurality of samples, for example, means for performing immunoassays at repeated intervals, which may extend over days, months, and years, for further studies as described above.

  The kit may further include means for collecting the sample. Means for collecting tissue samples from a subject are known in the art, for example, obtaining a skin sample such as a scissor or clipper to obtain a hair or nail sample, or a plastic stick or oral swab. A device for obtaining a fluid sample such as a lancet for generating a blood sample, or a hollow needle for aspirating CSF. Means for collecting environmental samples are also known in the art, such as, for example, a sealable liquid container for collecting a fluid sample. The means for collecting the sample may further comprise a means for storing the sample, for example, a liquid container (container) or solid substrate, a solid support, a multititer plate, a test tube, a tray, etc., wherein the storage is performed. The means may further include reagents for stabilizing and / or storing the sample.

  Kits of the invention may include means for sample preparation, as described elsewhere. The kit can be, for example, a means for mechanically separating the tissue sample, or chemically separating the tissue sample, using strong acids, enzymes, purifiers, etc., as described elsewhere in this disclosure. Means for preparing a tissue sample for analysis, such as means for carrying out the analysis. The means for sample preparation can include means for processing the sample to obtain different fractions, whereby protein-bound BMAA (eg, in protein fractionation) and free BMAA (eg, soluble Or in the cytoplasmic fraction) separately. Means for sample preparation may include means for total sample extract, and may include means for analyzing both protein-bound and free BMAA in the total sample extract. One of skill in the art will be able to prepare kits suitable for use in any particular immunoassay, and it will be understood that the precise physical embodiment may vary depending on the type of assay contemplated.

  A preferred kit is a commercial unit prepared to determine the presence of BMAA in a tissue sample from a subject. Another preferred kit is a commercial unit for determining the presence of BMAA in an environmental sample. The components of such a kit may include, for example, antibodies, microtiter plates, standard reagents, as well as various diluents and buffers, as described above. The kit may also contain a neurotoxic amino acid conjugate bound to a solid support, or an antibody bound to a solid support. The solid support is capable of diffusing or moving the sample applied to the microtiter plate, or material, along one or more of its dimensions, such as a "dipstick" or a permeable material. The surface may be a substance or the like, where the neurotoxic amino acid and its neurotoxic derivative bind to the antibody and form a detectable complex, while unbound substance passes through beads or the like in the column. The kit may also contain a labeled antibody or conjugate of one or more neurotoxic amino acids to be analyzed and neurotoxic derivatives thereof.

  According to one aspect, the kit comprises one or more compositions used to practice at least one method of the invention, packaged in a suitable packaging material. According to one aspect, the kit of the invention comprises a label and / or packaging insert for practicing at least one method of the invention. The term "packaging material" as used herein refers to the physical structure that contains the components of the kit. The packaging material can maintain the ingredients under sterile and / or stable conditions and can be composed of materials commonly used for such purposes (eg, paper, cardboard, glass, plastic, foil, ampules, etc.). . The terms "label" and "package insert" refer to appropriate instructions for practicing at least one method of the present invention. The instructions may be written in one or more languages, graphical instructions shown by figures, photographs, diagrams, etc., recorded verbal instructions, instructions coded on a fixed computer-readable medium, or the present invention. It should be understood that any other form of instruction suitable for conveying instructions for using the components of the kit to practice at least one method of the present invention may be used.

  In one embodiment, a kit of the invention comprises a label and / or package insert for detecting the presence of a neurotoxin in a subject or environmental sample by determining the presence of BMAA or a BMAA derivative. In one embodiment, the kit includes instructions for treating a subject in vitro, in vivo, or live in vitro. In an additional embodiment, the kit comprises a label or package insert containing instructions for treating the subject, in vivo, or live in vitro.

  The instructions can include instructions for practicing the immunoassays of the invention, as described herein. The instructions can be on a "print", for example, on paper or cardboard in the kit, on a label affixed to the kit or packaging material, or attached to a vial or tube containing the components of the kit. it can. The instructions may include audio or video tape, and optionally a disk (floppy diskette or hard disk), an optical CD such as a CD- or DVD-ROM / RAM, an electrical CD such as a magnetic tape, RAM and ROM. It can be included on a computer readable medium, such as a storage medium, a hybrid of these, such as a magnetic / optical storage medium.

  The kits of the present invention also include one or more detection means (eg, a detection enzyme and a detection enzyme substrate, or biotin and a biotin-binding moiety, a colloidal gold, a fluoriphore, Other labeling moieties, such as staining groups). The kit of the present invention can further include a buffer, a preservative, or a stabilizer. The kit can further include control components for preparing a standard curve and a calibration assay. Each component of the kit can be enclosed in a separate and distinct container. For example, a kit can include a single unit for detecting a neurotoxic amino acid, particularly BMAA, in a subject. Alternatively, the kit can include multiple units for detecting neurotoxic amino acids in multiple samples. Alternatively, the kit can include multiple units for detecting multiple neurotoxic amino acids in a single sample or in multiple samples. The components of the kit can be a mixture of one or more containers, and all of the various containers can be in single or multiple packages.

  In one embodiment, the kit comprises one or more compositions for detecting the presence of neurotoxicity in a subject by measuring BMAA or a BMAA derivative in a tissue sample, packaged in suitable packaging material. Including. In another embodiment, the kit comprises one or more compositions for detecting the presence of neurotoxicity in an environmental sample by measuring BMAA or a BMAA derivative, packaged in a suitable packaging material.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein.

Embodiment 1 FIG. Antiserum raised against BMAA Polyclonal antibodies capable of recognizing BMAA are produced by a method adapted from a protocol for producing and evaluating polyclonal antibodies against the cyanobacterial liver toxin microcystin-LR (Metcalf et al. 2000, Water Research 32: 2761-2767, Chu et al. 1989, Appl Environ Microbiol 55 (8): 1928-1933). Briefly, BMAA (molecular weight 118 Da) is conjugated to a macromolecule and introduced into a mammalian host by adapting the method for antibody ligation as disclosed in Harlow and Lane, Antibodies, A Laboratory Manual. When stimulated, the immune response was stimulated (Cold Spring Harbor Laboratory, 1988). The carboxyl and amine functions at the chiral center of BMAA were chosen for glutaraldehyde (GLU) and carbodiimide (EDC) linkers, respectively, and glutaraldehyde-BMAA (GLU-BMAA) and carbodiimide-BMAA (EDC-BMAA). Produced. GLU-BMAA and EDC-BMAA were conjugated to keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA), respectively, and the following BMAA complexes: BSA-GLU-BMAA (BGB), BSA-EDC-BMAA (BEB), KLH-EDC-BMAA (KEB), and KLH-GLU-BMAA (KGB).

  Specifically, a glutaraldehyde-bound BMAA complex was prepared as follows. A 5 mg / mL solution of BMAA was prepared by adding an equal volume of 2 × PBS to a 50 μL aliquot of BMAA (10 mg / mL in water). In KLH-GLU-BMAA (KGB), a solution of KLH was prepared at a concentration of 10 mg / mL in PBS. 40 μL of BMAA was added to 1 mL of KLH solution (10 mg / mL), followed by 960 μL of PBS. A 0.2% glutaraldehyde solution in PBS (about 25% stock) was prepared. An equal volume of glutaraldehyde was slowly added to the carrier protein BMAA solution with constant stirring and then incubated for 1 hour at room temperature. Glycine from a 1M stock in PBS (pH 7.4) was incubated with shaking for 1 hour to a final concentration of 200 mM. The KGB complex was separated from other reactions by dialysis against PBS (4 changes of 2 L overnight). After dialysis, the protein concentration of the solution containing the KLH complex was determined, and KGB was stored at -20 ° C in 500 μg aliquots.

  For BSA-GLU-BMAA (BGB), the same procedure was used, starting with a 10 mg / mL BSA solution. After dialysis, the protein concentration of the solution containing the BSA complex was determined and BGB was stored at -20 ° C in 500 μg aliquots.

  EDC-conjugated BMAA conjugate was prepared as follows. 50 μL of the BMAA stock (5 mg / mL) was added to the microcentrifuge tube. Prepare a solution of EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride in PBS at a concentration of 11.1 mg / mL, add 450 μL of the EDC solution to the BMAA solution, add 0.1 M The pH was adjusted with NaOH to pH 8. The mixture was incubated at room temperature for 5 minutes, the pH was checked and adjusted with NaOH if necessary.For KLH-EDC-BMAA (KEB), 10 mg / mL was added. A solution containing 1 mL of KLH carrier protein at a concentration was added to the EDC-BMAA solution and the mixture was incubated for 4 hours at room temperature.Sodium acetate (pH 4.2) was added to a final concentration of 100 mM (1 The reaction was stopped by adding 150 μL for a .1 M stock). The KEB complex was separated from the other reactions by dialysis against PBS (4 changes of 2 L overnight) After dialysis, the protein concentration of the solution containing the KLH complex was determined The KGB was then stored at −20 ° C. in 500 μg aliquots.

  For BSA-EDC-BMAA (BEB), the same procedure was used, starting with a 10 mg / mL BSA solution. The BEB complex is separated from the other reactants by dialysis as described above, the protein concentration of the solution containing the BSA complex is determined, and the BEB is stored in a 500 μg aliquot at −20 ° C. did.

  The KLH-BMAA complexes, KLH-EDC-BMAA (KEB) and KLH-GLU-BMAA (KGB), which were used in immunization to produce antisera, were selected. The BSA-BMAA conjugates, BSA-EDC-BMAA (BEB) and BSA-GLU-BMAA (BGB), were used to coat anti-sera and coat immunoassay plates to develop immunoassays.

  New Zealand White rabbits were injected with KLH-EDC-BMAA (KEB) or KLH-GLU-BMAA (KGB) according to standard protocols (Metcalf et al., 2000). Briefly, rabbits received a first subcutaneous injection of a solution containing BMAA-KLH complex (KEB or KGB) and complete Freund's adjuvant, and two weeks later received an intravenous booster injection. Additional antigen booster injections were given at monthly intervals, and one week after each booster injection, there was a serum harvest (about 20 mL of blood). Harvested blood was allowed to clot and stored overnight before separating serum from red blood cells. The isolated serum was subjected to three ammonium sulfate precipitations before dialysis against PBS. Aliquots (100 μL) of each serum sample, including pre-immune serum (“null serum”), were stored at −20 ° C. until needed. One rabbit was immunized with KGB and sera were harvested at eight different time points (eighth "bleed"). A total of two rabbits were immunized with KEB, the first rabbit died after the second blood draw, the second rabbit was then immunized with KEB, and the serum was 5% from the second rabbit. Harvested at different times (fifth "blood draw"). To distinguish results using sera from two rabbits immunized with KEB, the first rabbit immunized with KEB was identified as the "KLH-EDC1-B MAA" antiserum and immunized with KEB. Serum from the second rabbit given was identified as the "KLH-EDC2-BMAA" antiserum.

Embodiment 2. FIG. Preliminary Characterization of Antisera Generated Against BMAA Preliminary measurements showed that the immune sera contained antibodies that reacted with BMAA in addition to the antibody reaction with carrier proteins and crosslinkers.

Antibody Capture Immunoassay As described above using an antibody capture immunoassay (Metcalf et al., 2000, Chual. 1989) similar to that shown to successfully characterize antisera raised against microcystin. The antiserum raised against the BMAA conjugate, prepared as described above, was characterized. Here, the binding of the rabbit antibody was detected using an anti-rabbit secondary antibody labeled with horseradish peroxidase (HRP) and the chromogenic synthetic HRP substrate 3,3 ′, 5,5′-tetramethylbenzidine (TMB). . After the wells were coated with BMAA conjugate (100 μL per well), the plates were washed and blocked with 1% (w / v) dry milk powder in PBS (Marvel brand, 180 μL per well). After blocking, the plate was further washed and then incubated at 37 ° C. for 1 hour before applying the primary antibody (in PBS). After addition and incubation of the primary antibody, each plate was washed and goat anti-rabbit IgG-HRP (Sigma) in PBS was added to the wells (100 μL per well) at a dilution of 1/10000. The plate was then incubated at 37 ° C. for 1 hour and then washed. HRP substrate TMB was added to each well (100 μL per well) and the plate was developed for 30 minutes at room temperature. The HRP-TMB reaction was stopped by adding 100 μL of 1 M HCl, and the absorbance at ₄₅₀ nm (A ₄₅₀ ) of each plate was measured to determine the amount of bound antibody in each well.

Here, the BSA-BMAA complex was coated on the wells of the immunoassay plate at various coating concentrations, and a sample of the antiserum produced against the BSA-BMAA complex prepared by the same method was loaded onto the immunoassay plate. (I.e., the antiserum raised against BEB was added to the BEB-coated wells and the pattern was the same for BGB), and the antibody was added to the BSA-BMAA complex coated on the plate. captured by binding to, wherein the antibody binding, goat anti-rabbit IgG-HRP secondary antibody, using the measurement of _{a 450} relative TMB substrate, and each well was measured. BMAA-coated wells were also probed with null serum from rabbits prior to immunization with the BMAA complex used to coat the wells, where the null serum was purified as described above. did.

  Using a different experimental approach to antibody capture immunoassay, BMAA was bound directly to the surface of the wells (ie, not through BSA or KLH complex) and anti-BMAA antibody binding was measured as described above. The effect of pH and plate format on antibody capture involves BMAA bound directly to various types of multiwell plastic plates known to have different binding properties at different pH values using solutions with different pH values. And measured. BMAA (20 μg / mL) was dissolved in buffers with different pH values as follows. Acetate at pH 4, PBS at pH 7.4, carbonate at pH 9.6. The BMAA solution was then coated on Nunc brand MAXISORP ™, Nunc brand MEDISORP ™, and Nunc brand MULTISORP ™ plates (Thermo Fisher Scientific) on plates known to have pH specific binding properties. An aliquot of the antiserum raised against the BMAA complex, or null serum (pre-immune serum), was then tested at a dilution of 1/1000 for each serum sample for each plate format / pH combination, respectively. In the designed design, it was added to each well. The following serum samples were tested for binding to different plate formats at different pH values. Two null serum samples from two different rabbits (NS1, NS2); two antisera raised against KLH-EDC-BMAA in different rabbits (EDC1-1 were isolated before rabbit death). 1st blood draw from one BMAA-EDC immunized rabbit, EDC2-1 was the first blood draw from a 2nd BMAA-EDC immunized rabbit); and 1 month interval from the same rabbit Four consecutive bleeds of antisera raised against KLH-GLU-BMAA (Glu1, Glu2, Glu3, Glu4) collected in the above. The format with BMAA bound directly to the surface of the wells resulted in significantly higher absorbance readings in each well, where antiserum was added to the BMAA complex and the added null serum (NS) was added. Compared to the measured absorbance of the wells with, this format was not used in further studies.

Verification Specific to the BMAA Portion of the BMAA Complex The BMAA portion of the BMAA complex used to verify the presence of BMAA-specific antibodies, ie, to elicit an immune response to produce antisera To verify the presence of reactive antibodies, antisera raised against the BMAA complex was tested. Here, antisera raised against the KLH-BMAA conjugate was tested for its ability to bind to the BSA-BMAA conjugate synthesized using the “reverse” cross-linking chemistry. Since the rabbit antiserum raised against the KLH-conjugated immunogen does not have an antibody against BSA, a BMAA-specific antibody was detected using the BSA-BMAA conjugate. Furthermore, it was understood that as a result of conjugating BMAA to the carrier protein (KLH) using different cross-linking chemicals (GLU, EDC), a change in the conformation of the composition of the BMAA molecule could be expected. Therefore, antisera raised against BMAA conjugated via one cross-linking chemistry may show low reactivity to BMAA conjugated via "reverse" cross-linking chemistry Was expected.

  When the antisera raised against the KLH-BMAA conjugate was tested against the BSA-BMAA conjugate prepared with both cross-linking chemistries, each antiserum showed "reverse" cross-linking. A positive reaction was shown for BSA-BMAA conjugated via the above chemicals. That is, the antiserum produced against the EDC-conjugated BMAA complex (KEB) showed a positive reaction against GLU-linked BSA-BMAA (BGB). Similarly, antisera raised against the GLU-linked BMAA complex (KGB) showed a positive reaction against EDC-conjugated BSA-BMAA (BEB). As expected, each antiserum tested positive for BSA-BMAA conjugated via the same cross-linking chemistry as the BMAA conjugate used to generate the antiserum. It was noted that antisera raised against KGB appeared to react more strongly to EDC cross-linked samples compared to antisera raised to KEB reactive to GLU cross-linked samples. . The first animal immunized with KEB was given an antiserum (KLH-EDC1-BMAA) that produced a better response than the antiserum of the second animal immunized with the complex (KLH-EDC2-BMAA). Generated.

  These results indicate that BMAA-specific antibodies were present in the IgG pool of harvested antisera and that the cross-linking method used for conjugation had no detectable negative effect on the ability of the antisera to recognize BMAA. That was shown.

Antiserum reactivity with free BMAA determined using indirect competitive ELISA The ability of antisera raised against the BMAA complex to bind free BMAA was determined by indirect modification from the antibody capture immunoassay described above. Determined using a competitive ELISA format. That is, the antibody capture immunoassay was performed on plates with BSA-BMAA conjugate binding to the wells, except that free BMAA (unbound and unconjugated) and antiserum were added simultaneously to each well. Free BMAA and bound BSA-BMAA complex on the wells were generally performed as described above to compete for antibody binding.

Briefly, each assay well was coated by adding 100 μL of BSA-BMAA (BGB or BEB) in PBS at pH 7.4, 1 hour at 37 ° C., 2 μg / mL, 1 μg / mL, Alternatively, incubation was performed using a BSA-BMAA concentration of 0.5 μg / mL. The wells were then blocked with 1% (w / v) dry milk powder in PBS (Marvel brand, 180 μL per well). “Primary antibody” containing free BMAA (50 μL / well L-BMAA, 10 μg / mL in MilliQ water) and antiserum to KLH-conjugated BMAA (anti-KEB or anti-KGB, 50 μL / well diluted in PBS) Solutions were added to each well using antisera at dilutions of 1/1000, 1/5000, 1/10000, 1 / 50,000, and 1 / 1,000,000 (ie, 1/1 × 10 ⁶ ). Antibodies that bind to BSA-BMAA coated on the wells are detected at 1 / 10,000 using goat anti-rabbit IgG-HRP (Sigma) in PBS (100 μL / well), washed and washed with TMB substrate ( KPL Laboratories, 100 μL / well) was added. The HRP / TMB reaction was then stopped by adding 100 μL / well of 1 M H ₂ SO ₄ and the amount of bound antibody was determined by measuring the absorbance at ₄₅₀ nm (A ₄₅₀ ). Antibody binding, i.e., the ability of free BMAA to compete with BSA-BMAA complexes for binding to antigen (free BMAA) were reported as calculated% _{B 0} value as the ratio of _{A 450} values using the following equation .

% B ₀ = (absorbance of test sample / absorbance of control) × 100
"Control" values were measured in wells to which no free BMAA was added. A% B ₀ value of less than 100 (<100) indicates that in the test sample, a portion of the antibody bound to free BMAA in solution and the amount of antibody binding to the BSA-BMAA complex coated on the wells Decreased by. In other words, the ₀ value% _B of <100, antibodies in the antisera to detect free BMAA, and showed that the bound.

  Preliminary evaluations were coated with antiserum raised against the KLH-BMAA conjugate, and BSA-BMAA conjugate with the same and different cross-linking chemistry as the KLH-BMAA conjugate Performed using wells. Assays using the same cross-linking chemistry were (A) anti-KGB antiserum (GLU AS) from the third blood draw added to BGB coated wells, and (B) added to BEB coated wells. This was performed using anti-KEB antiserum (EDC2 AS) from a second bleed of EDC rabbit 2 obtained. Assays with different cross-linking chemistries were added to (A) anti-KGB antiserum (GLU AS) from the third blood draw added to BEB-coated wells, and (B) BGB-coated wells. This was performed with anti-KEB antiserum (EDC2 AS) from a second bleed of EDC rabbit 2. Free BMAA, antiserum dilution, coating concentration, and reaction conditions were as described above.

In the assay using the same cross-linking chemistry, little binding to free BMAA was detected except for an antiserum raised against KGB (GLU AS) at a dilution of 1/1000. There is a% B ₀ value of 98% in wells coated with 2 μg / mL BGB, and at a dilution of 1 / 50,000, the antiserum raised against KEB (EDC2 AS) is 2 μg / mL. It had ₀ value 80%% B in the wells coated with BEB but reproducibility been a problem.

Assays using different cross-linking chemistries improved detection of binding to free BMAA. A% B ₀ value of <100% was measured in most of the assays, and the antibodies in the antiserum bound to free BMAA in solution instead of binding to the coated BSA-BMAA complex on assay plate wells To do so. Antisera raised against KGB (GLU AS) had a 80% lower% _{B 0} value in coated wells in BEB. Antiserum raised against KEB (EDC2 AS) had a low% B ₀ value of 70% in BGB coated wells. These results indicated that the antiserum raised against the KLH-BMAA complex was able to detect free BMAA, ie, the antiserum contained an antibody that specifically reacted with free BMAA. .

Purification of antiserum by immunoprecipitation with KLH After the above experiments showed that the antiserum raised against the KLH-BMAA complex was able to detect free BMAA, the purification procedure was followed. Developed to remove foreign elements such as antibodies to KLH carrier proteins and cross-linking molecules. The hapten of interest is cross-linked to the carrier protein, and the hapten-crosslinker-carrier protein conjugate is used for immunization, and the mammalian immune system uses all of the complex, including the cross-linking molecule and the carrier protein. It is expected that antibodies can be produced against this portion. Therefore, it is often considered effective to perform an additional purification step to remove or reduce antibodies to non-hapten epitopes, thereby providing anti-hapten antibody in the preparation of antisera. Increase the relative abundance of A preferred method involves immunoprecipitation or use of an immunoaffinity column.

  Immunoprecipitation with KLH was performed to remove antibodies to KLH as follows. An aliquot of 1 μg KLH is added to the stabilized antiserum preparation (antibody solution), the mixture is allowed to react at 37 ° C. for 30 minutes, the mixture is centrifuged and a fresh aliquot of 1 μg KLH is used. The supernatant was transferred to an unused tube for subsequent immunoprecipitation. At each immunoprecipitation, one aliquot of the antiserum was removed and tested for reactivity to KLH and BSA-BMAA complex. The results of KLH immunoprecipitation with both antisera (ie, antisera raised against GLU-linked BMAA (KGB) and antisera raised against EDC-conjugated BMAA (KEB)) indicated that antibodies to KLH Can be removed and the reactivity to the BSA-BMAA complex was shown to be maintained in the partially purified “KLH-cleaned” antiserum.

  Fifteen rounds of immunoprecipitation with KLH were performed with a third bleed (GLU AS) in the antiserum raised against KGB, and after each round of immunoprecipitation, aliquots were taken and at different concentrations. Reactivity to KLH and, at different concentrations, to BSA-BMAA with the same cross-linking chemical, ie, BGB, were tested.

  Fifteen rounds of immunoprecipitation with KLH were performed, and a second bleed (EDC2 AS) of a second EDC rabbit in antiserum raised against KGB was performed, and an aliquot was taken after each round of immunoprecipitation And tested at different concentrations for reactivity to KLH and at different concentrations for BSA-BMAA with the same cross-linking chemical, ie, BEB. For both antisera, results of tests of reactivity to KLH and BSA-BMAA showed that multiple rounds of immunoprecipitation were able to remove antibodies to KLH, but were partially purified. The antiserum showed a stable level of reactivity to the BSA-BMAA complex.

After 15 rounds of immunoprecipitation with KLH, each partially purified antiserum ("KLH-purified antiserum") was tested for reactivity to free BMAA in solution (1 μg / mL), The assay wells used were coated with BSA-BMAA conjugates with the same or different cross-linking chemistry, and the indirect competitive ELISA format described above for testing for reactivity with free BMAA was used. In this procedure, the diluted antiserum was (1) BSA-BMAA with the same cross-linking chemical used to conjugate KLH-BMAA used to produce the antiserum. The conjugate, and (2) coated with a BSA-BMAA conjugate of a cross-linking chemical opposite to the cross-linking chemistry used to conjugate KLH-BMAA used to generate antiserum Tested with free BMAA at 1 μg / mL in wells. 1 / 1000-1 / at 1 × 10 ⁶ dilution were tested antisera. Partially purified antisera, compared to controls, as shown by the 80% to 100% of the% _{B 0} values were detect free BMAA. Although it was determined that the partially purified antiserum was able to react with free BMAA, it was further determined that the partially purified antiserum had a higher affinity for the BMAA complex. Was done. Partially purified antisera, compared to controls, with a ₀ value 80% to 100% of% _B, showed reactivity to free BMAA. It was further determined that the partially purified antiserum had a higher affinity for the BMAA complex than for free (unconjugated) BMAA.

Embodiment 3 FIG. Reactivity of anti-BMAA antiserum with BSA complex of structurally similar amino acids Since BMAA is a derivative of alanine and also has a structure similar to glutamic acid, the antiserum produced against BMAA is Experiments were performed to determine if they exhibited reactivity with alanine and BSA-glutamic acid conjugate. The antisera KGB and KEB produced against the non-immunoprecipitated ("normal") and partially purified ("KLH-purified") BMAA conjugates were combined with the BSA-alanine and BSA-glutamic acid conjugates. Was tested for reactivity. GLU-linked and EDC-linked complexes with BSA having alanine and glutamic acid were prepared and tested as follows. BSA-GLU-alanine (BGA), BSA-EDC-alanine (BEA), BSA-GLU-glutamic acid (BGG), and BSA-EDC-glutamic acid (BEG).

  Serial dilutions of "normal" and KLH-purified antisera were prepared as described above (before immunoprecipitation with KLH, "normal" antisera were obtained and KLH-purified antisera were rounded off for 15 rounds). Anti-serum was added to the wells coated with various BSA amino acid conjugates, and antibody binding was measured using the ELISA format described above. By doing so, the reactivity to each complex (BGA, BEA, BGG, BEG) was tested.

  Both normal and KLH-purified antisera showed some reactivity with BSA-glutamate and BSA-alanine complexes, but different patterns of reactivity were dependent on the cross-linking chemistry. It was found to be different.

  When the same cross-linking chemical was used in the KLH conjugate used to generate antisera and in the BSA conjugate used to test antisera, it was cleared with both "normal" and KLH The antisera showed reactivity with the BSA-alanine and BSA-glutamic acid conjugates in addition to the expected reactivity with the BSA-BMAA conjugate. Samples of antisera raised against both normal and KLU-cleared KGB (ie, GLU-linked BMAA) are more sensitive to GLU-linked BMAA complexes than to GLU-linked alanine or GLU-linked glutamate complexes. It had higher affinity. In contrast, antisera to both normal and KLH-cleared KEB (ie, EDC-conjugated BMAA) were recognized equally for all three EDC-conjugated complexes.

  In contrast, when the reverse cross-linking chemistry is used in the KLH conjugate used to raise the antiserum, the BSA conjugate will react with the antiserum (eg, reactive with BEB, BEA, and BEG). Anti-KGB antiserum tested for), and the antiserum showed little reactivity with any of the conjugates.

Since the antisera showed reactivity with the (recognized) BSA-amino acid conjugate with the same cross-linking chemistry, indirect competition was used to test these combinations for reactivity with free BMAA. Used in ELISA format. That is, an indirect competition ELISA was used to measure the ability of free BMAA to compete for antibody binding in wells coated with the BSA-amino acid complex. For each antiserum, the BSA-amino acid conjugate was cross-linked using the same cross-linking chemistry as the KLH-BMAA conjugate used to generate the antiserum. Therefore, antisera raised against normal and KLH-purified KGB were tested for reactivity with free BMAA (1 μg / mL) in BGB, BGA, or BGG-coated wells. Normal and KLH-cleared antisera to KGB were tested for reactivity with free BMAA (1 μg / mL) in wells coated with BEB, BEA, or BEG. The chemicals both crosslinking, normal (not immunoprecipitated) antiserum at% B ₀ value of 80% to 100%, it was possible to detect free BMAA in solution. However, anti-serum purified with KLH as well as normal (non-immunoprecipitated) anti-serum to detect free BMAA in solution was not performed, but free BMAA was detected in some assays.

Further purification by immunoprecipitation with BSA-alanine Of the three BSA-amino acid complexes tested (BSA-BMAA, BSA-alanine, BSA-glutamic acid), of both cross-linking chemicals (BGA, BEA) The BSA-alanine conjugate showed the lowest reactivity with antisera. Thus, the KLH-purified antiserum was further purified by immunoprecipitation with BSA-alanine to create a highly enriched antiserum preparation with antibodies specific for BMAA only. Then, KLH-purified antiserum prepared as described above (after 15 rounds of immunoprecipitation, a third blood collection of antiserum produced against KGB; after 15 rounds of immunoprecipitation using KLH, A second bleed of the second EDC rabbit for the antiserum produced against it) was subjected to an additional 14 rounds of immunoprecipitation with BSA-alanine. After each immunoprecipitation (IP1-IP14), antiserum was added to wells coated with various BSA-amino acid conjugates, and antibody binding was measured using the ELISA format described above. The reactivity with the BSA-BMAA complex, the BSA-alanine complex, and the BSA-glutamic acid complex was tested.

  At a dilution of 1/1000 (the most concentrated solution tested), after immunoprecipitation with BGA, the antiserum against KGB has good reactivity against BGB, and against other amino acid complexes BGA and BGG. It showed low reactivity, indicating that the antiserum was specific for BMAA. Reactivity with BGB was reduced during the first 8 rounds of immunoprecipitation 1-8 (IP1-IP8) and maintained a good level of reactivity during the final 6 rounds of immunoprecipitation (IP9-IP14). However, reactivity with BGA and BGG continued to decline with continued rounds of immunoprecipitation. The reactivity trend observed in this experiment suggested that antibodies recognizing the GLU crosslinker had been removed by immunoprecipitation with BGA.

  At a dilution of 1/1000, antisera to KEB immunoprecipitated with BEA showed a decrease in reactivity with all BSA-amino acid complexes when immunoprecipitation was continued. This result is consistent with the results reported above, which indicate that antisera raised against KEB to recognize specific BSA-amino acid complexes (both "normal" and purified with KLH) Hardly any difference in performance.

  After immunoprecipitation with BSA-alanine, the antisera were tested for their reactivity with free BMAA at 1 μg / mL using the indirect competitive ELISA format described above. Both immunoprecipitated antisera (antisera to KGB and antisera to KEB) showed little reactivity with free BMAA.

Embodiment 4. FIG. Testing Specificity and Cross-Reactivity of Anti-BMAA Antiserum Using Glutaraldehyde-Linked Coating Format Antiserum produced as described above was used to convert amino acids and other haptens into microtiter plates for enzyme immunoassay. An alternative method for coating was tested using an adaptation of the immunoassay method of Ordronneau et al. (1991). Et al. Disclose that the method described above used an assay substrate, wherein the carrier protein was conjugated to the substrate and the amino acids or haptens were conjugated to the carrier protein, which was reproducible and accurate. Inconsistencies and problems. Have developed an immunoassay for glutamate (Glu), in which Glu is linked directly to the plastic surface via glutaraldehyde instead of being conjugated to a carrier protein, and a Glu-coated immunoassay plate is used for Glu. It was used to test antisera produced against it.

  The method of Ordronneau et al. Was used to prepare immunoassay plates coated with glutaraldehyde-linked BMAA (not carrier protein) for testing antisera of the present invention. A third "bleed" of antiserum raised against KGB was used for the experiment. The method of Ordronneau et al. Used MAXIMARP ™ and MULTISORP ™ plates coated with BMAA and glutamic acid, and anti-BMAA antiserum (anti-KGB, third blood draw), from 1/1000 to 1/100, Various dilutions of 000 were performed in the presence of BMAA, at concentrations of 0-1 mM, or in the presence of glutamic acid, at concentrations of 1 nM-1 mM. MULTISORP ™ plates showed higher absorbance values, as well as better glutaraldehyde binding and subsequent BMAA binding. At dilutions of 1/1000, 1/2000, 1/5000, and 1 / 10,000, the antisera raised against KGB are higher when BMAA is added to the plate at high coating concentrations (100 μM and 1 mM). It showed increased binding to BMAA coated plates, which resulted in increased BMAA coated plates and was presumed to be usable for antibody binding. No effect on antibody binding was seen with increasing concentrations of glutamate.

  Antisera raised against KGB at dilutions of 1/1000 and 1/2000 (anti-KGB antiserum) were tested for cross-reactivity with BMAA and other amino acids to test specificity for BMAA. . The plates were coupled via glutaraldehyde linkage to BMAA, L-alanine (L-Ala), L-glutamine (L-Gln), L-tyrosine (L-Tyr), glycyl-glycine (glygly), L-glycine ( L-Gly), L-leucine (L-leu), L-phenylalanine (L-Phe), gamma-aminobutyric acid (GABA), L-glutamic acid (L-Glu), and L-aspartic acid (L-Asp) Were coated at coating concentrations of 0.2 mM, 0.5 mM, 1 mM, and 10 mM. As shown in FIG. 3, at both dilutions (1/1000 and 1/2000), the anti-KGB antiserum showed strong recognition of BMAA and showed little cross-reactivity with the other amino acids tested. At a dilution of 1/1000, recognition of BMAA by the anti-KGB antiserum (FIG. 3A) increased at BMAA coating concentrations of 0.1-10 mM, ie, the intensity of the signal increased with increasing BMAA binding. . At a dilution of 1/2000, the recognition of BMAA by the anti-KGB antiserum (FIG. 3B) reaches a plateau at a BMAA coating concentration of 1 mM, whereby saturation binding is reached at that concentration. Was shown.

  In this immunoassay format, at both dilutions, the anti-KGB antiserum showed strong recognition of BMAA and little cross-reactivity with the other amino acids tested, but at 1/1000, the anti-KGB antiserum was In particular, at 10 mM coating, it showed little reactivity with L-glycine and glycyl-glycine (FIG. 3A), which indicates that L-glycine does not eliminate any residual glutaraldehyde groups that may be present on the carrier protein. Not all were unexpected when used during the procedure of immunogenic conjugates cross-linked with glutaraldehyde to activate. Of all the other amino acids tested, only the anti-KGB antiserum showed slight reactivity with GABA at 10 mM and with aspartic acid at 0.2 mM. Upon binding to glutaraldehyde, the structural changes in these molecules affect subsequent recognition by the antibody, and it is determined whether further testing for `` free '' amino acids may be necessary to confirm these findings. Did not.

Embodiment 5 FIG. Specificity of anti-BMAA antiserum to BMAA from different sources; Determination of reactivity of isomer specificity The immunization and immunoassay described above were performed using Sigma (now Sigma-Aldrich Inc .; Cat. No. B107, lot number). 097H4746) using commercially available BMAA. The immunoassay was performed using two new different batches of BMAA: a batch from an unused lot of BMAA commercially available from Sigma (Lot 065K4707), and a batch of synthetic BMAA obtained from Peter Nunn at the University of Portsmouth, UK. And went again. Immunoassay using unused batches of BMAA from each of the new batches, i.e., BMAA from Sigma (Sigma-Aldrich, Lot 065K4707), and synthetic BMAA from Peter Nunn (University of Portsmouth, UK). Using glutaraldehyde capture (a glutaraldehyde-linked antibody capture form as described above) to determine the ability of various antisera to bind to various targets, such as BMAA from different batches, Ran.

Briefly, the wells of a Nunc MULTISORP ™ plate were washed with distilled water. Each well received 100 μL of 0.5% glutaraldehyde in 100 mM NaH ₂ PO ₄ (pH 4.5) and the plates were incubated at 37 ° C. for 1 hour. The plate was washed twice with 180 μL of 100 mM NaH ₂ PO ₄ (pH 4.5) (ie, each well of the plate was washed). A 100 μL aliquot of a target, eg, BMAA, prepared in 100 mM Na ₂ HPO ₄ (pH 8) was added to each well and the plate was incubated at 37 ° C. for 1 hour. The plate was washed three times with 180 μL of 100 mM Na ₂ HPO ₄ (pH 8) for each wash. A 100 μL aliquot of 0.1 M ethanolamine, prepared in 100 mM Na ₂ HPO ₄ (pH 8), was added to each well and the plate was incubated at 37 ° C. for 1 hour. Plates were washed three times with 0.05% Tween 20 / PBS (PBST) in each wash. A 180 μL aliquot of 1% “Marvel” brand dry milk powder in PBS was added to each well and incubated at 37 ° C. for 1 hour. Plates were washed three times with PBST. A dilution of the primary antibody in PBS was prepared and 100 μL of the (diluted) primary antibody was added to each well. Plates were incubated at 37 ° C. for 1 hour. Plates were washed three times with PBST. For detection, 100 μL of IgG-HRP (1/10000, Sigma goat anti-rabbit IgG-HRP) was added to each well and the plate was incubated at 37 ° C. for 1 hour. Plates were washed three times with PBST. For quantification, the HRP synthetic chromogenic substrate TMB was added (100 μL per well), allowing the color to develop for 30 minutes at room temperature. The reaction was stopped by adding 100 μL of 1 M H ₂ SO ₄ and the absorbance at 450 nm was measured for each well of the plate.

  In one experiment, a third bleed (KBG3) of antiserum raised against KGB (anti-KGB antiserum) was performed using KLH by precipitation with ammonium sulfate and addition of 10 μg KLH to 100 μL antiserum. Cleared using ELISA or in a further round of KLH immunoprecipitation by immunoprecipitation, incubation at 37 ° C. for 30 minutes, and centrifugation at 2000 × g for 5 minutes, collection of the supernatant. KLH-purified anti-KGB antiserum at dilutions of 1/1000, 1/2000, 1/4000, 1/8000, 1/16000, and 1/32000 was used to provide 2% of BMAA at a BMAA coating concentration of 1 μM to 5 mM. Glutaraldehyde-linked BMAA from each of the three new batches was added to the plate and antibody binding to the plate was measured. For both BMAA batches, the anti-KGB antiserum showed an increase in signal intensity (reactivity) with increasing BMAA coating concentrations, which indicated that the anti-KGB antiserum was specific for BMAA. It was confirmed that it contained a specific antibody. The immunoassay used here had a detection limit of 10 μM BMAA and the maximum reactivity (maximum absorbance) was measured at a BMAA coating concentration of 0.5 mM for both BMAA batches.

  The signal intensity (reactivity) of different antiserum dilutions for each BMAA coating concentration was determined for each of the two different batches of BMAA, and the correlation coefficient was plotted on the y-axis as P.E. Calculated from a plot of values from an unused lot of BMAA from Sigma on the x-axis against values from synthetic BMAA from Nunn. A separate regression analysis was performed and correlation coefficients were calculated for 1/1000, 1/200, and 1/4000 dilutions of the anti-KGB antiserum. The correlation coefficient showed a positive correlation (> 0.89) between the different BMAA batches. However, the slope of the regression line for each anti-KGB antiserum dilution indicates that the signal obtained using the same antiserum dilution and the same BMAA coating concentration is P.A. It was shown to be twice as high for an unused lot of BMAA from Sigma as compared to the signal obtained with synthetic BMAA from Nunn (University of Portsmouth, UK). The 1/1000 dilution has a regression slope of 0.65, the 1/2000 dilution has a regression slope of 0.56, and the 1/4000 dilution has a slope of 0.4. It has a regression line slope of 49.

It should be noted that BMAA from all sources was a synthetic product, but each product was a composition of different isomers. The synthetic BMAA from Sigma lot number 097H4746, used as the original antigen for conjugation and immunization, was described by the manufacturer if it contained more than 94% of the L isomer. BMAA, supplied by Peter Nunn (University of Portsmouth, UK), was described as containing a mixture of the D and L forms, in approximately equal amounts, with a slight preference for the L isomer (Peter Nunn). , Personal communication). The results from the regression analysis (see above) were evaluated in terms of the different isomers of each product and these results were
The predominantly L-isomer (> 94% L-isomer) antiserum produced against KGB prepared with BMAA from lot number 097H4746 is reduced to glutaraldehyde-trapped L-BMAA isomer. It showed preferential binding and showed little reactivity with the D isomer of BMAA. Under these conditions, the antiserum bound to the L isomer of BMAA and did not substantially bind to the D isomer of BMAA.

  After the isomer-specific reactivity of the antiserum raised against unpurified KGB ("normal" antiserum) was demonstrated, the reactivity of the normal anti-KGB antiserum was partially purified. A comparison was made between the "KLH-purified" anti-KGB antiserum and the partially purified "alanine-purified" anti-KEB antiserum. All antisera were used to test binding to BMAA in wells at a coating concentration of 1 μM to 5 mM at dilutions of 1/1000 and 1/2000, with free BMAA present at 500 μM. Both unpurified "normal" anti-KGB antiserum and partially purified KLH-purified anti-KGB antiserum were coated with BMAA with increasing BMAA coating concentration of BMAA up to 0.5 mM. It showed increased binding to the plate. Unpurified "normal" anti-KGB antiserum at both dilutions (1/1000 and 1/2000) showed a slight decrease in binding at a coating concentration of about 0.5 mM BMAA. Anti-KGB antiserum purified with both dilutions of KLH shows a plateau of binding at a coating concentration of 0.5-5 mM BMAA, which is limited at coating concentrations of BMAA above about 0.5 mM. Antibody accessibility and / or binding saturation of the antibody. In contrast, at a dilution of 1/1000 and 1/2000, the alanine-cleared anti-KEB antiserum was detected on BMAA (ie, a plate coated with BMAA) at any coating concentration of 1 μM to 5 mM BMAA. No possible binding was shown.

Unpurified "normal" anti-KGB antiserum (third bleed) was determined for its ability to bind free BMAA using a glutaraldehyde capture immunoassay, modified for the indirect competitive binding assay described above. Tested to Test wells were coated with BMAA coating concentrations linked with 50 μM, 200 μM, 500 μM, 1 mM, and 5 mM BMAA glutaraldehyde. At a concentration of 500 μM, free BMAA and normal anti-KGB antiserum (third bleed) at 1/1000, 1/2000, and 1/4000 dilutions were added to the test wells and glutaraldehyde in the wells was added. To determine antibody binding to ligated BMAA and to determine the reactivity of the antiserum with free BMAA,% _B0 values were calculated as described above. In this experiment, the antiserum was able to detect (react with) free BMAA (ie,% B ₀ <100) in most assays. Results,% B ₀ values, with increasing BMAA coating concentrations, or with increasing antiserum concentration (low antiserum dilution), considered to be reduced for showed general trend. The maximum% B ₀ value measured was 56%, indicating a 44% reduction in antibody binding to BMAA coated on wells due to antibody binding to free BMAA.

Embodiment 6 FIG. Amplification System for Detecting Anti-BMAA Antibody Binding The above experiments demonstrate that the antiserum raised against the BMAA complex has apparent isomer-specific reactivity with L-BMAA and other amino acids. Constructed to contain antibodies with little cross-reactivity with, antiserum could be used to detect free BMAA at a concentration of about 500 μM (59 μg / mL). As described below, experiments were performed to evaluate various amplification systems for the signal of free BMAA and their ability to improve detectability without requiring further purification of the antisera. .

Amplification of Anti-BMAA Antibody Signal Immunoassay sensitivity was determined by the VECTASTAIN ™ ABC-peroxidase kit (VECTASTAIN ™ ABC for rabbit IgG) to generate horseradish peroxidase (HRP) detection complex with more detection enzymes. Elite kit, Cat.No.PK-6101, Vector Laboratories, Burlingame CA), which results in stronger coloration (more intense signal), and HRP-conjugated IgG (IgG. -HRP) resulted in higher absorbance values compared to the standard assay with (HRP). By using the VECTASTAIN ™ system with increasing BMAA coating concentration in an antibody capture immunoassay, when compared to the signal measured with a standard IgG-HRP, as described in the experiments above, Significantly strong (increased) signal measurements were possible. However, the background signal was also significantly enhanced by the VECTASTAIN ™ system, which required the development of appropriate controls to use in assessing antisera.

The VECTASTAIN ™ system was used to measure the effects of different glutaraldehyde concentrations, different free BMAA concentrations, and different antiserum dilutions on the ability of unpurified normal anti-KGB antiserum to detect free BMAA. Used in the indirect competition assay format in the glutaraldehyde linked antibody capture assay described above. The two glutaraldehyde concentrations tested for effect on the BMAA coating were 0.2% glutaraldehyde and 0.5% glutaraldehyde. Wells were coated with BMAA through glutaraldehyde ligation using BMAA coating solutions of 100 mM, 50 mM, and 20 mM BMAA. A third bleed of antisera raised against KGB (KGB3) was used at concentrations of 1/8000, 1/16000, and 1/20000. Free BMAA was added to wells at concentrations of 1 μg / mL and 10 μg / mL, and control wells had no added free BMAA. In the design of this experiment, antisera at 1/8000 was tested for reactivity with both levels of free BMAA, ie, this experiment involved incubating antisera at 1/8000 with 1 μg / mL free BMAA. And at 1/8000 the antiserum was incubated with 10 μg / mL free BMAA. At a dilution of 1/16000 and 1/20000, the antiserum was incubated only with 10 μg / mL of free BMAA. The VECTASTAIN ™ amplification system was used as described above to amplify the results. % _B0 values were calculated with or without blank correction.

The results from all experimental designs show that the anti-KGB antiserum contained antibodies that reacted with free BMAA, ie,% B ₀ <100, antibodies bound to free BMAA covered the wells. It did not bind to glutaraldehyde linked BMAA. The effect of free BMAA was most pronounced against antisera at dilutions of 1/16000 and 1/20000 when incubated with 10 μg / mL free BMAA. At both glutaraldehyde concentrations tested for their effect on BMAA coating wells (ie, 0.2% and 0.5% glutaraldehyde), the assay was free at concentrations of 1 μg / mL and 10 μg / mL. BMAA is both before and after correcting the ₀ value% B for blank, indicating that could be detected by an immunoassay.

Further Modifications to Alter Immunoassay Sensitivity The standard immunoassay system described above, which showed unpurified “normal” anti-KGB antiserum, was able to detect free BMAA at 59 μg / mL and used the VECTASTAIN ™ amplification system. Use provided improved detection. Further developments and modifications were performed to further improve the sensitivity of potential BMAA immunoassays, especially chemical modifications such as biotinylation.

  Amplification of biotin-avidin in combination with VECTASTAIN The biotin-avidin reaction is one of the known maximum affinity reactions, where a biotinylated probe is rapidly and specifically attached to an enzyme or solid phase using an avidin system. Can be done. Biotinylated BMAA probes were produced and used in combination with the avidin-HRP conjugate from the VECTASTAIN ™ kit to improve the sensitivity and specificity of the BMAA immunoassay, as described below.

Biotin-BMAA reactivity with anti-BMAA antiserum using direct ELISA Biotinylated BMAA was tested using ELISA designs where wells were coated with antiserum at various dilutions and varying amounts of biotinylated BMAA Was added to the wells and the binding of biotinylated BMAA to the immobilized antibody was measured. The purpose of the assay was to determine whether the biotin-BMAA complex was feasible for use in a BMAA immunoassay and to determine whether the biotinylated BMAA bound to antisera raised against the BMAA complex. As such, the ELISA was a direct antibody binding assay and did not use free BMAA.

  Briefly, antisera raised against KEB and KGB were coated on assay plates, and at various dilutions, a biotin-BMAA probe was added to the wells coated with the antiserum, and the Biotin-BMAA binding to the immobilized antibody was measured using a chromogenic marker, such as avidin conjugated to HRP.

  Biotinylated BMAA was prepared as follows. A solution of BMAA in PBS was prepared at a concentration of 1.18 mg / mL. Biotin (with linker) solution was prepared with enough EZ-link Sulfo-NHS-LC-LC-Biotin to make a 6.99 mg / mL solution in PBS. For mixing and labeling, 1 mL of BMAA was mixed with 1 mL of biotin solution and the components were allowed to react at room temperature before use in the ELISA. A stock solution of 1 M BMAA-biotin (biotinylated BMAA) was prepared and diluted on a volume basis. At 1M, a stock solution of biotin-BMAA was added to 1/100 (0.01M), 1/500 (0.002M), 1/1000 (0.001M), 1/5000 (0.0002M), 1/10000. (0.0001M), 1 / 50,000 (0.00002M), and 1/100000 (0.00001M) (vol / vol) dilutions.

  The following antisera were used. Anti-KEB antiserum, sixth blood draw from a second rabbit immunized with KEB (EDC6, sixth blood draw from rabbit KLH-EDC2-BMAA); anti-KGB antiserum, ninth blood draw (GLU9). Each antiserum represented a single harvest (a single "blood draw") and was subjected to an initial partial purification by ammonium sulfate precipitation. Antisera were diluted 1/1000, 1/5000, and 1/10000 in PBS prior to use in the ELISA, as described below.

For ELISA, wells were coated with antiserum by adding a 100 μL aliquot of diluted antiserum and incubating the plate at 37 ° C. for 1 hour. Plates were washed three times with PBST. The wells of the plate were blocked with 180 μL of 1% Marvel dry milk powder in PBS and the plate was incubated at 37 ° C. for 1 hour. The plate was then washed three times with PBST before adding the biotin-BMAA complex to the plate. Plates were incubated at 37 ° C. for 1 hour before washing three times with PBST. A 100 μL aliquot of the avidin-HRP conjugate supplied using the VECTASTAIN ™ amplification kit was added to each well and then incubated at 37 ° C. for 1 hour. Plates were washed three times with PBST before adding TMB substrate (100 μL per well) for 30 minutes at room temperature. The reaction was stopped by adding 1 M H ₂ SO ₄ (100 μL) and the absorbance at 450 nm was measured for each well.

BMAA binding to immobilized antibody (antiserum) showed strong signal dose response, decrease in signal intensity _(indicating antibody binding to A 450 BMAA- biotin probes), the "dose" of BMAA- biotin probe When it fell, it fell. At all dilutions (1/1000, 1/5000, and 1/10000), both antisera (anti-KEB and anti-KGB) were associated with a decrease in BMAA-biotin (ie, an increase in BMAA-biotin dilution). Showed the same signal dose pattern with decreasing signal intensity.

Different detection probes: single (non-amplified) avidin-HRP probe In another experiment, a different commercially available single (non-amplified) HRP-avidin probe was added to the VECTASTAIN ™ HRP-avidin conjugate. As an alternative to use, it was used to detect BMAA-biotin probe bound to immobilized antibody from anti-KGB antiserum. The ELISA was performed as described above. Briefly, anti-KGB antiserum (ninth bleed, see above) was coated on assay plates at a dilution of 1/1000 and, as described above, from 1/100 (0.01 M) to 1/100. At a dilution of 100,000 (0.00001 M), biotin-BMAA was added to the wells. A single commercially available HRP-avidin probe (Sigma-Aldrich, Cat. No. 1-3151, 250 μg / mL) was tested for its ability to detect binding of biotin-BMAA to the immobilized antibody. Avidin-HRP was diluted to provide different strength solutions: avidin-HRP diluted (vol / vol), 1/1000, 1/2000, 1/4000, and 1/6000. Each biotin-BMAA concentration was measured using a respective dilution of avidin-HRP.

  A strong signal dose response was observed with a single avidin-HRP probe. For each biotin-BMAA concentration (dilution), the strongest signal was seen in the assay with the HRP-avidin probe at a dilution of 1/1000, ie at the maximum concentration of the HRP-avidin probe. For each biotin-BMAA dilution, the signal intensity decreased with HRP-avidin concentration, ie, the signal decreased as the HRP-avidin probe was gradually diluted.

Reactivity of anti-BMAA antiserum with free BMAA in the presence of biotin-BMAA Assay wells were coated with biotin-BMAA at different dilutions and a single commercially available HRP-avidin probe was added at 5 μg / different dilution. At a concentration of mL, used in an indirect competitive ELISA format to detect the reactivity of anti-KGB and anti-KEB antisera with free BMAA. Both anti-KGB and anti-KEB antisera showed reactivity with 5 μg / mL free BMAA, with the strongest response as low as 78%, as evidenced by measurements of% B ₀ <100. Found in anti-KGB antiserum with ₀ value.

Embodiment 7 FIG. Use of anti-BMAA antibodies to detect BSA-BMAA complex in immunoblots BMAA can be associated with peptides and proteins in various ways, including physical attachment or association of the peptide to the surface, and It should be understood that BMAA incorporation into the polypeptide chain is involved. The experiments described above show that antisera raised against the KLH-BMAA complex (anti-KGB and anti-KEB antisera) recognize BMAA in complex and free form (ie, complexed BMAA and free BMAA). Contains possible antibodies. Therefore, as described above, antisera raised against the BMAA complex was used to detect BMAA association with the polypeptide in an immunoblot (Western blot). In one experiment, antisera probed immunoblots (Western blots) of various protein preparations and used these antisera to determine if they could recognize protein-associated BMAA.

  As demonstrated in the above experiments, anti-KGB and anti-KEB antisera were able to recognize the BMAA-BSA complex. Therefore, anti-KGB and anti-KEB antisera were used to probe Western blots of BSA and various BSA-BMAA complexes. The following samples were subjected to SDS gel electrophoresis and transferred to cell membranes for Western blot (immunoblot) analysis. BSA-GLU-BMAA (BGB), BSA-EDC-BMAA (BEB), and uncomplexed BSA (natural protein). Results from immunoblots using antisera raised against BMH conjugated with KLH to probe blots of the BSA-BMAA complex were chemically bound to the surface of high molecular weight proteins (eg, BSA) in the immunoblot. It showed promising signs for the detection of (complexed) BMAA.

  Proteins were loaded on polyacrylamide gels (10 μg protein per lane) and 4% stacking gels at 200 V for about 40 minutes using BioRad Mini-PROTEAN® II (BioRad, Hercules CA). Then, it was subjected to electrophoresis through a 12% separation gel.

  Proteins were transferred from polyacrylamide gels to nitrocellulose membranes overnight at room temperature (BioRad Mini Trans-Blot®, BioRad, Hercules CA) as follows. Transfer buffer (3.03 g Tris, 14.4 g glycine, 200 mL methanol; made up to IL using water) was prepared and stored at 4 ° C. Each nitrocellulose membrane was cut to fit the size of the gel on which the protein was transferred. Prior to transfer, all components were pre-wetted and equilibrated by immersion in gel, nitrocellulose membrane, filter paper, and fiber pad in transfer buffer. Open the "sandwich" by opening the holder cassette with the outside (gray) on the cleaning surface, place a pre-moistened fiber pad on the gray side of the cassette, place a piece of filter paper on the fiber pad and remove air bubbles Carefully place the pre-moistened nitrocellulose membrane on the gel, take care to remove air bubbles, place the filter paper on the nitrocellulose membrane, finally add the fiber pad and close the holder cassette Prepared. After adding a cooling unit and completely filling the tank with transfer buffer, transfer was performed at 30 V, 90 mA overnight (about 18 hours). After the transfer was considered complete, the quality of the transfer and the location of the protein bands could be visualized by reversible staining with Ponceau S. Cell membranes were marked as needed during this step.

For immunoblot analysis, cell membranes were removed from the transfer unit (or, where appropriate, from the Ponceau S destaining solution) and incubated in 0.1% dry milk powder (Marvel brand) / PBST for 1 hour. Incubated. The cell membrane was then washed 3 times (3 × 5) with PBST for 5 minutes per wash. If necessary, the nitrocellulose membrane was cut into strips corresponding to the sample lanes. Cell membranes were incubated with the primary antibody at various dilutions for 2 hours and then washed 3 times (3 × 5) with PBST for 5 minutes per wash for 5 minutes. In the experiments described herein, the ninth bleed of anti-KGB antiserum (GLU9 AS) and the sixth bleed of the second EDC rabbit of anti-KEB antiserum (EDC6 AS) were 1/100, 1 / 200 and 1/500 dilutions were used as primary antibodies. For secondary antibody labeling, cell membranes were incubated with IgG-HRP (1: 250) for 2 hours, then with PBST, 3 times (3 × 5) for 5 minutes per wash for 5 minutes. , Washed. The peroxidase substrate was prepared by mixing a solution of 15 mg of 4-chloronaphthal in 5 mL of cold methanol with a solution of 15 μL of H ₂ O _{2 in} 25 mL of PBS. To visualize antibody binding, a chromogenic peroxidase substrate was prepared by mixing the two solutions together and applying to the washed cell membrane. The reaction was monitored as the band developed (usually about 5-10 minutes). Further development was stopped by adding water. The cell membrane (whole cell membrane and / or strip) was then blot dried.

  Results from Ponceau S staining of nitrocellulose membrane to visualize all transferred BSA-containing proteins were compared to immunoblot (Western blot) results showing antibody binding to the transferred protein on the same cell membrane. . Results from Western blots showed similarities and differences from Ponceau blots. When the anti-KGB antiserum (9th bleed, Glu9 AS) was used as the primary antibody to probe the blot, the full strength of the antiserum preparation (1/100, 1/200, and 1/500 dilution) ) Appeared to react with the BGB sample (FIG. 4, lanes 1 of blots A, B, and C), while the BEB sample (lane 2 of FIG. 4, blots A, B, and C), or native BSA ( It was not considered to react with FIG. 4, lanes 3) of blots A, B and C. The BGB complex using anti-KGB antiserum showed antibody staining consistent with Ponceau staining previously observed for the samples, where the BGB samples had positions corresponding to 191, 85, and 70 kDa ( FIG. 4, lanes 1) of blots A, B, and C showed strong staining of the band. Lack of reactivity with the native BSA control indicates that the reactivity of the anti-KGB antiserum was specific for BMAA and / or GLU crosslinker and was not specific for BSA. The lack of reactivity with the complex cross-linked with EDC (BSA-EDC-BMAA, BEB) was due to the results of the aforementioned immunoassay that the anti-KGB antiserum was able to recognize BEB (see above). ), Difficult to interpret, suggesting that anti-KGB antiserum could be expected to recognize an epitope on BEB on Western blots.

  When the anti-KEB antiserum (6th bleed, EDC6 AS) was used as the primary antibody to probe the blot, the full strength of the antiserum preparation (1/100, 1/200, 1/500) was Reactive with BEB (FIG. 4, lanes 2 of blots D, E, and F) and BGB sample (lane 3 of FIG. 4, blots D, E, and F), the staining and strongly staining bands observed through the gel were , 191, 167, 60, 53, 35, 29, 21, and 10 kDa. At a dilution of 1/100, the anti-KEB antiserum showed little reaction with native BSA (FIG. 4, blot D, lane 1), but at dilutions of 1/200 and 1/500 (FIG. 4, blot E, No reaction was seen in lane 1 and blot F, lane 1). The reactivity of the anti-KEB antiserum with both BSA-BMAA conjugates (BEB and BGB) indicates that the anti-KEB antiserum was able to recognize both BEB and BGB, according to previous ELISA results. (See above).

Embodiment 8 FIG. Immunoblot analysis of cyanobacterial protein preparation from Kirindrospermopsis rhakiborskii strain CR3 Immunoblot analysis was performed on cyanobacterial protein preparation from Kirindrospermopsis rhakiborskii strain CR3, which included BMAA (protein It has previously been known to contain large amounts of cytoplasmic BMAA (free BMAA) in addition to (bound BMAA) (Cox et al. (2005) Proc Natl Acad Sci USA 102: 5074-5078). Kirindrospermopsis Rakiborskii strain CR3 ("CR3") was harvested from large scale cultures on the University of Dundee and prepared as follows. A sample of 175 mL of late log phase culture of filamentous cyanobacteria was removed and the gas vesicles were disrupted by mechanical shock (bombarding a packed centrifuge tube on a workbench). Was. The filament was centrifuged at 3500 rpm for 10 minutes (Heraeus Labofuge 400). The supernatant was removed and the pellet was resuspended and transferred to a 1.5 mL microcentrifuge tube at 4000 rpm for further centrifugation (2.5 minutes, Eppendorf centrifuge 5415D). The supernatant was removed again and the pellet was resuspended in 50 mM Tris buffer at pH 7.5 to a final volume of 1 mL. The suspension was sonicated on ice for about 1 minute to lyse the cells and release the protein. The suspension was centrifuged again and the protein concentration of the suspension was analyzed using dye-bound protein (Sigma) by measuring the absorbance at 595 nm (Bradford, 1976, Anal Biochem 72: 248-254). The suspension was then modified by adding EDTA to a final concentration of 1 mM and glycerol to a concentration of 10% (v / v).

  Prior to electrophoresis and immunoblot analysis, a sample of the CR3 protein preparation was used to prepare a sample to test whether BMAA reacts with any proteins present in the cyanobacterial protein preparation. Were pre-incubated with free BMAA (BMAA was "loaded"). Native BSA was used as a control.

  SDS-PAGE was performed as described above, using 28 μg protein per lane (28 μg CR3 whole protein extract), using 4% stacking and 12% separation gel, and transferred to nitrocellulose membrane. Was. After the protein was transferred to the nitrocellulose membrane, anti-KGB antiserum (9th blood draw, KGB9) and anti-KEB antiserum (6th blood draw, KEB6) were used to probe the primary antibody to probe the Western blot of CR3 protein. Used as As shown in FIG. 5, both anti-KGB and anti-KEB antisera reacted with one or more epitopes on the protein in the CR3 protein preparation (lane 2 of all blots). Both antisera reacted with the CR3 protein, but with different reaction profiles. The anti-KGB antiserum reacted with proteins having a molecular weight in the range of 10-120 kDa. The anti-KEB antiserum reacted with a protein having a molecular weight in the range of 21-196 kDa. Preincubation of the CR3 protein preparation with free BMAA ("addition") had no detectable effect on antiserum reactivity (lane 3 of all blots). These results indicated that the CR3 cyanobacterial protein preparation contained a protein with an epitope recognized by the anti-BMAA antiserum.

  The BSA control showed slight reactivity with both antisera at the highest antiserum dilution tested (1/100), but this reactivity was seen when the antiserum concentration was reduced. Did not. The results showed that when the primary antibody was present at high concentrations, some showed non-specific binding to BSA (FIG. 5, lane 1 of all blots).

Embodiment 9 FIG. Testing for non-specific reactivity in immunoblots To test the possibility that some of the reactivity observed on immunoblots (see above) was due to non-specific reactivity, immunoblot analysis was performed. Was performed with antisera raised against low dilutions of BMAA (anti-KGB and anti-KEB), and null (pre-immune) sera (FIG. 6). Trials of Kirindrospermopsis rhakiborskii strain CR3 ("CR3") extract and native BSA were evaluated for non-specific reactivity. For SDS-PAGE, 28 μg of protein (CR3 total protein extract) or 10 μg of BSA was loaded in each lane and the gel composition, running conditions, transfer conditions, and immunoblot conditions were as described above. Was.

  Anti-KGB antiserum (KGB9) was used as the primary antibody at dilutions of 1/200, 1/500, 1/1000, and 1/2000. Anti-KEB antiserum (EDC6) was used as the primary antibody at dilutions of 1/200, 1/500, 1/1000, and 1/2000. Null serum (NS) collected from rabbits prior to immunization was used as the primary antibody at a dilution of 1/200.

  Both antisera showed reactivity with the protein and negligible reactivity with the BSA sample in the CR3 extract at all dilutions. A comparison of the reactivity patterns seen for the anti-KGB and anti-KEB antisera showed that the anti-KEB antiserum reacted with a number of protein bands and that the staining of these bands was more pronounced, while the anti-KGB The antiserum was considered to react with only one protein complex. The anti-KEB antiserum reacted with the CR3 protein at all dilutions in a region with a distinct band visible on the blot, corresponding to an average molecular weight of about 66 kDa. At a dilution of 1/200, the anti-KEB antiserum showed reactivity with BSA in the region corresponding to a molecular weight of about 54-66 kDa. The anti-KGB antiserum showed strong reactivity with the CR3 protein in the region corresponding to an average molecular weight of about 50 kDa. Null serum also showed reactivity with CR3 protein in the same region, corresponding to an average molecular weight of about 50 kDa.

  In the CR3 extract, NS reacted with a band at 50-60 kDa (lanes 2, 12). However, when comparing the staining intensities, it was evident that the anti-KGB antiserum showed significantly higher staining intensity and recognized many protein bands in the CR3 sample. This difference was also seen when comparing anti-KEB antiserum and null serum (NS), but the contrast is less dramatic. In this experiment, null serum (NS) was used in one animal, as previous experiments with null serum did not show differences between null sera taken from different rabbits prior to immunization with different BMAA conjugates. Recovered from rabbits, there was no prior indication of any rabbit-specific reactivity with BMAA or BMAA complex. From these results, null serum (NS) was used as a control indicator for Western blots, as if the band began to appear on cell membranes incubated with null serum (NS) control, the color reaction was stopped. That is, if the band starts to appear in the sample probed with NS, then the color reaction is complete, since the specific reaction is probably complete and it is understood that any further coloration is probably due to a non-specific reaction. To stop.

  The anti-KEB antiserum is believed to react with a wide variety of CR3 protein bands, and since the band was more defined than the anti-KGB reactive protein, the anti-KEB antiserum was further diluted than the anti-KGB antiserum in the assay described below, It was determined that antisera could be used. Furthermore, because more concentrated antiserum solutions (eg, 1/100 dilution) were thought to result in increased non-specific binding, lower concentrations of primary antibody (higher dilutions) required specific detection of BMAA-containing proteins. Was used in the analysis described below to improve the likelihood of

Embodiment 10 FIG. Immunoblot analysis of protein preparations from other cyanobacterial strains Protein extracts were further analyzed for immunoblot analysis using anti-KGB and anti-KEB antisera to compare protein profiles and antiserum reactivity. Prepared from cyanobacterial strains. Total protein extracts were prepared from Microcystis strain PCC7820, Spirulina strain PCC8005, and Baltic Nodularia. A protein preparation from Kirindrospermopsis Rakiborskii strain CR3 ("CR3") was included as a comparative analysis and a positive control. Samples from each strain were loaded onto a gel for SDS-PAGE (29 μg protein / lane), gel composition (eg, 4% stacking, 12% separation), running conditions, metastasis conditions, and immunity Blot conditions were as described above.

  The SDS-PAGE gel stained the protein using Coomassie staining to show the protein profile for each cyanobacterial protein preparation. When the SDS gel was stained with the more sensitive silver nitrate stain and compared to the less sensitive Coomassie blue, an additional protein band was seen, which was not seen with the Coomassie stain, but with an immunoblot (Western blot). The extracts showed the presence of various proteins in the extract that were potentially detectable by analysis. After protein transfer from the SDS-PAGE gel to nitrocellulose membrane, protein transfer was assessed by reversibly staining cell membranes with Ponceau S to visualize proteins. SDS-PAGE gels were tested for protein before and after transfer to confirm that the protein was present in the gel (before) and transferred from the gel to a nitrocellulose membrane (after).

  Blots of the four cyanobacterial protein extracts were used to determine if antisera raised against the BMAA complex reacted with the protein in these extracts, and that any particular cyanobacterial protein was , Were probed with anti-KEB and anti-KGB antisera to indirectly investigate whether they were thought to be associated with BMAA. Blots of the four cyanobacterial protein extracts were also probed with null serum (NS) to test for non-specific reactivity. The staining patterns and results from the protein strains and the immunoblots were compared.

  When immunoblots probed with anti-BMAA antiserum were compared to immunoblots probed with null serum, the responses seen with anti-BMAA antiserum showed a different pattern and higher signal intensity. The strength of the anti-BMAA antiserum response in the cyanobacterial protein preparation was different for each strain, with Kirindrospermopsis rhakiborskii CR3 showing the strongest (darkest) response and Microcystis PCC7820 the next strongest, Then, Spirulina PCC8005, the weakest (pale color) reaction, was seen with Baltic nodularia. When the immunoblot was probed with null serum, a band corresponding to a protein with a molecular weight of approximately 59 kDa was observed. When the immunoblots were probed with anti-BMAA antiserum, bands were labeled in each strain as follows. For CR3, the bands corresponding to proteins having molecular weights of about 243, 149, 129, and 114 KDa were labeled, and the "coated samples" corresponding to proteins having molecular weights of about 42-104 kDa were labeled. For PCC8005, bands corresponding to proteins with molecular weights of approximately 249, 129, 44, and 29 kDa were labeled. For Baltic nodularia, bands corresponding to proteins with molecular weights of 136, 123, 44, and 30 kDa were labeled. For PCC7820, a "coated sample" corresponding to a protein having a molecular weight of about 69-106 kDa was labeled.

Embodiment 11 FIG. Reactivity of anti-BMAA antiserum with commercially available organisms Cyanobacteria showed strain-specific differences in the reactivity of anti-BMAA antisera (see above), so other organisms were compared to anti-BMAA antiserum. To elucidate their potential reactivity, they were evaluated by immunoblot analysis. Commercial dietary supplements of baker's yeast (Saccharomyces cerevisiae) and dietary supplements of "green algae" (Chlorella sp.) Were tested for their reactivity with anti-BMAA antisera by immunoblotting. The protein preparation from the chlorella dietary supplement reacted strongly with the anti-BMAA antiserum, while the baker's yeast (Saccharomyces cerevisiae) preparation showed little reactivity.

Embodiment 12 FIG. Reactivity of anti-BMAA antiserum with E. coli, Tetracermis, and Chlorella Further studies were performed using pure strains with a known history, as the origin of the commercial products tested above could not be established. In addition, these pure strains of an organism were tested as potential "negative controls" for comparison with cyanobacteria. To better understand the context of possible BMAA association with cyanobacteria, E. coli (strain HK29; Dr. H.K. Young, University of Dundee), green alga Chlorella vulgaris and green alga Tetracermis Pure strains of the species were obtained as possible negative controls.

  Pure strains of E. coli (strain HK29), Chlorella vulgaris, and Tetracermis species were obtained, harvested, and total protein extracts prepared. Samples from each strain were loaded onto a gel for SDS-PAGE (29 μg protein / lane), gel composition (eg, 4% stacking, 12% separation), running conditions, metastasis conditions, and immunity Blot conditions were as described above. A protein preparation of Kirindrospermopsis Rakiborskii CR3 was included for comparison.

  As shown in FIG. 7, the expected pattern of reactivity with the CR3 protein sample is seen, and protein samples from other organisms show some reactivity with the antiserum used to probe the blot. showed that. 1/500 or 1/1000, anti-KEB antiserum or 1/500 (dilution tested only), anti-KGB antiserum can detect any of green algae, chlorella, and tetracermis proteins Did not show any significant reactivity.

  At a dilution of 1/500, the anti-KEB antiserum (EDC6 AS) labeled the bands in the CR3 sample corresponding to proteins with molecular weights of approximately 124, 89, 59, and 35 kDa (FIG. 7, lane 6) At a dilution of 1/1000, the anti-KEB antiserum labeled a band in CR3 corresponding to a protein with a molecular weight of approximately 121, 94, 79 kDa (FIG. 7, lane 12). At 1/500, the anti-KEB antiserum has a molecular weight of about 124, 97, 86, 79, 73, 59, 50, 46, 38, 35, 27, 24, 22, 16, 12, 11, and 9 kDa Strong labeling of the band in the E. coli sample corresponding to the protein was shown (FIG. 7, lane 7). At a dilution of 1/1000, the anti-KEB antiserum corresponds to proteins with molecular weights of about 109, 98, 88, 61, 48, 43, 38, 27, 25, 23, 16, 14, 13, and 9 kDa. Although the band in the E. coli sample was weakly labeled, the band was labeled at 1/500, much lower intensity than that with antiserum (FIG. 7, lane 13).

  At a dilution of 1/500, the anti-KGB antiserum was labeled in a band in the "coated" CR3 sample corresponding to proteins with molecular weights in the range of about 84-36 kDa (Figure 7, lane 16). At a dilution of 1/500, the anti-KGB antiserum showed strong labeling of the band in E. coli samples corresponding to proteins with molecular weights of about 66, 58, 49, 44, and 24 kDa (FIG. 7, lanes 17).

  Null serum (at a dilution of 1/500) showed little reactivity with CR3 (FIG. 7, lane 2). In E. coli samples, the null sera was labeled with bands corresponding to proteins having molecular weights of about 91, 13, and 12 kDa (Figure 7, lane 3). In Chlorella, the null serum labeled a band corresponding to a protein having a molecular weight of about 11 kDa (FIG. 7, lane 4). In tetraselmis, the null serum labeled a band corresponding to a protein having a molecular weight of about 10 kDa (FIG. 7, lane 5).

  The reactivity of the anti-KEB and anti-KGB antisera with proteins of E. coli strain HK29 indicates that the reactivity of any of the previously tested cyanobacterial strains with the same amount of total protein extract was loaded in each lane. More potent than serum reactivity. Extensive specific protein bands in E. coli preparations were stained by both antisera. A sample of E. coli strain HK29 was harvested and freeze-dried for BMAA analysis by HPLC.

  Because denatured SDS-PAGE was used to separate proteins from all organisms tested above, and that different protein bands labeled with anti-KGB and anti-KEB antisera on immunoblots were isolated from different organisms. Have been isolated and denatured, so that antisera raised against the BMAA complex, as evidenced by analysis of reactivity with the synthetic immune complex, and protein extracts from living organisms The above experiments suggested that BMAA was incorporated into the polypeptide chain, as evidenced by the results showing that it did.

  Various modifications may be made to the preferred embodiments without departing from the spirit and scope of the invention, as defined in the appended claims.

Claims (15)

specifically binds to beta-N-methylamino -L- alanine (BMAA) L isomer (L-BMAA), but shows the cross-reactivity of less than 25% with respect to D-isomer of BMAA, An antibody for distinguishing between L-BMAA and the D isomer of BMAA (D-BMAA), comprising L-alanine, L-glutamine, L-tyrosine, glycyl-glycine, L-glycine, L-leucine, L- An antibody that exhibits less than 25% cross-reactivity with phenylalanine, gamma-aminobutyric acid (GABA), L-glutamic acid, or L-aspartic acid.   2. The antibody of claim 1, wherein said antibody binds free L-BMAA.   The antibody of claim 1, wherein said antibody binds to protein-bound L-BMAA.   2. The antibody of claim 1, wherein the antibody binds to both free L-BMAA and protein-bound L-BMAA.   The antibody according to claim 1, wherein said antibody is a polyclonal antibody.   The antibody according to claim 1, wherein said antibody is a monoclonal antibody.   The antibody of claim 1, wherein said antibody is an antibody fragment. 2. The antibody of claim 1, wherein said antibody is detectably labeled.   2. The antibody of claim 1, wherein said antibody is labeled for use in an in vivo diagnostic imaging method.   A kit for screening a sample and detecting the presence of the β-N-methylamino-L-alanine (BMAA) L isomer, comprising at least one container, wherein the at least one container is A kit comprising the antibody according to 1.   The kit of claim 10, wherein the antibody that binds to L-BMAA is detectably labeled.   The kit according to claim 11, further comprising a composition for detecting the labeled antibody bound to the sample.   The kit of claim 10, wherein the antibody that binds to L-BMAA is unlabeled.   14. The kit of claim 13, further comprising a container containing a labeled secondary antibody that binds to the unlabeled antibody. The kit of claim 10, further comprising a container containing a control sample containing a known amount of L-BMAA.